Bums (1992) 18,(6), 486-489
486
Printed in Great Britain
Are plasma endotoxin levels related to burn size and prognosis? S. Endo’, K. Inadaz, M. Kikuchi’, Y. Yamadal, T. Kasoi’, M. Kondo*, S. Hoshi’, H. Yamashitaz, M. Suzuki2 and M. Yoshida’ ‘Critical Care and Emergency Morioka, Japan
Center and ‘Department
of Bacteriology,
Plasma endofoxin concentrations were determined in 42 pafienfs with burns covering more than 20 per cent of the body surface area, using the endofoxin-specific Endospecy assay and freafmenf of plasma by a new method developed by ourselves. The normal endofoxin level was 9.8pglml or less. In the early period affer injury when no infection was present, very few patients had an endofoxin level above 9.8pglml and endofoxin levels did not correlate with the area of fhe bums or wifh prognosis. However, later in the clinical course, endofoxin levels were correlated sigr#icunfly with the burned area and with the prognosis.
Introduction Along with recent progress in both medical and surgical management, the results achieved in the treatment of bums have been improved markedly. However, infection still remains a major problem during bums treatment. It is thought that endotoxin derived from Gram-negative bacteria has a role in various complications which burns patients develop (Horn et al., 1984; Mason et al., 1986). There are reports of endotoxin entering the blood from the intestinal tract in the early stages of bum injury, and as the burned area becomes larger the blood endotoxin level apparently increases (Woodruff et al., 1973; Deitch and Rodney, 1987; Winchurch et al., 1987; Marek et al., 1989). The conventional assay methods were not specific for endotoxin, but an endotoxin-specific assay (Endospecy) was developed several years ago (Obayashi et al., 1985). Moreover, it was almost impossible to measure accurately the plasma endotoxin level after pretreatment of plasma samples by the conventional perchloric acid method, so we developed a new method for the pretreatment of plasma (Takahashi, 1988; Inada et al., 1991). This new pretreatment method made it possible to obtain fairly accurate plasma endotoxin levels using the Endospecy test. In this study, we monitored plasma endotoxin levels in bums patients from the time of admission and investigated the relationship of endotoxin to bum size and clinical outcome.
Materials and methods Patients and blood collection The subjects were 42 patients with bums affecting not less than 20 per cent of their body surface area (BSA) who could 0 1992 Butterworth-Heineman 030554179/92/060486-04
Ltd
School of Medicine,
Iwate Medical University,
undergo successive blood tests from early after their admission. They included 24 males and 18 females aged from 7 to 87 years (mean f s.d., 47.5 f 23.7 years). The bum size ranged from 20 to 95 per cent BSA (45.5 f 21.5 per cent). The subjects were divided into three groups according to the burned area: group A, 20-39 per cent BSA (26.8 f 6.3 per cent, n = 18); group B, 40-59 per cent BSA (47.4 f 4.7 per cent, n = 13); and group C 2 60 per cent BSA (76.8 f 13.2 per cent, n = 11). There were 25 survivors and 17 patients died. In all patients, blood collection was started within 2 h after injury. Blood samples were taken every 4 h in the first 24 h after admission, every 8 h in the period from 24 to 72 h, and every 12 h in the period from 72 h to 1 week. After 1 week, blood was taken once a day, unless disseminated intravascular coagulation (DIC) or septic shock was observed, in which case it was taken every 2-3 h. Blood collection was continued until death or until no risk of infection remained. Blood was collected into heparinized tubes and centrifuged at 3000 r.p.m. for 40s to obtain plasma, which was stored at - 80°C until assayed. Endotoxin assay The plasma endotoxin level was measured using an endotoxin-specific chromogenic limulus test, the Endospecy test (Seikagaku Corporation, Tokyo, Japan). Plasma was pretreated by the new perchloric acid method (new PCA method) to remove factors interfering with the limulus test. The accuracy of measurement of endotoxin after the new PCA pretreatment method was improved about eight-fold (Takahashi, 1988; Inada et al., 1991) when compared to conventional PCA pretreatment of plasma (Obayashi et al., 1985). When using the conventional PCA method, the precipitate formed after PCA addition is discarded and the supematant is assayed for endotoxin. However, most of the plasma endotoxin activity is actually contained in the discarded precipitate (Inada et al., 1990). Accordingly, we devised a new PCA treatment method which allowed this endotoxin to be retained. Brieftly, 100 pl of 0.18 N NaOH was added to a 100 pl sample of test plasma and incubated at 37”C, 5 min. Then 100 ~1 of 0.32 M PCA was added and further incubation was performed at 3 7°C for 10 min. The precipitate was then dissolved in 200 pl of 0.18 N NaOH by vigorous mixing with a vortex mixer. Finally, 50 pl of the
Endo et al.: Plasma endotoxin levels in burned patients
487
solution was transferred to another tube, and 50 ~1 of 0.2 M Tris-HCl (pH 8.0) was added. Thus, the plasma sample was diluted tenfold. An aliquot (100 ~1) of this diluted plasma was then used for the Endospecy limulus test. This test amoebocyte lysate obtained from the utilizes Japanese horseshoe crab, Tachypletls tridents&. For quantitative measurement, a synthetic chromogenic substrate (n-tertbutoxycarbonyl-L-leucy-L-glycyl-L-argininep-nitroaniline) was added to the lysate to act as the substrate for the clotting enzyme. A 100 ~1 aliquot of Endospecy was added to 100 ~1 of treated plasma, mixed for several seconds and then incubated at 37°C for 30 min. The amount of P-nitroaniline (pNA) released from the substrate was detected after diazo-coupling. The absorbance of the solution was measured at 545 nm, and a standard curve was plotted on a bilogarithmic scale using lipopolysaccaride solution (E. cd 0111:B4, Difco). The normal endotoxin level as determined by this method was less than 9.8 pg/ml (Inada et al., 1991). With the Endospecy test, false-positive reactions are sometimes observed in patients receiving sulpha drug therapy. This is due to the presence of an aromatic primary amine in their chemical structure. The false-positive value was obtained by incubating treated samples with water instead of Endospecy, and the true value was then calculated by subtracting the false-positive value from the Endospecy test value. The research protocol was approved by the Institutional Review Board of Iwate Medical University and all the patients gave their informed consent to participation. Statistical
analysis
Data are expressed as the mean & s.d. Statistical analysis was performed with the two-tailed Wilcoxon rank-sum test for unpaired data and the level of significance was set at P< 0.05.
patients remained under 9.8pg/ml in this first week (Figttre I), and no significant difference was observed between them. In addition, no significant difference was noted when plasma endotoxin levels were reviewed on a daily basis. When a comparison was made between the survivors and those who died with respect to plasma endotoxin levels in the first week after admission, no significant difference was observed (F@re 2). Similarly, no significant difference was observed in the daily changes of the plasma endotoxin levels. When the total clinical course was considered, the number of patients with plasma endotoxin levels above 9.8 pg/ml was 5/18 (27.8 per cent) in group A, 11113 (84.6 per cent) in group B and 7/11 (63.3 per cent) in group C (Figure 3). However, if the four patients who died within 48 h were excluded, all seven remaining patients in group C had raised endotoxin levels. The plasma endotoxin levels in groups A, B and C were 44.2 f 142.9, 160.1& 225.2 and 236.6 & 282.5 pg/ml, respectively, and no significant difference was observed among the three groups. However, when the four patients who died early were excluded from group C, the level rose to 391.0 rt 269.8 pg/ml, and this was
5
:: ;; D : E Mean
Results
Time
Only one patient had a plasma endotoxin level above 9.8 pg/ml at the time of admission. In the first week after the bum injury, only five patients (6/956 blood samples, 0.70 per cent) showed plasma endotoxin levels over 9.8pg/ml. The range of elevation was 11.6 pg/ml (10.8 f 0.6 pg/ml).
The mean plasma endotoxin
not
great,
being
postinjury
(days)
V-PC ,--P
o.oL
< 0.05
-NS-
Y-N-Y--~
.
. .Cl
10 2 E _
_________________ _________ ____-- _________
? s.d.
Figure 2. Changes in the plasma endotoxin levels from the time of injury to I week later in survivors (0 -0, n=25) and non-survivors ( l 0, n = 17) (excluding four patients dying within 48 h).
10.2-
levels of all three groups of
.8
10 r-__-----_________________--______---_._.____--_..~------__----~
E -En ,a a, -2 .G
:
.
___ __________________ 9. * .
1
-
P
0
z
Mean
E; 0
0
1
2 Time
/ 3 postinjury
, 5
/ 4
i I 6
s.d. 7
_;_
(days)
Figure 1. Changes in the plasma endotoxin level from the time of injury to I week later (maximum daily levels in relation to bum size as per cent BSA). O0, 20 per cent < TBSA < 40 per cent (n = 18); l 0, 40 per cent d TBSA < 60 per cent (VI= 13); A ---A, 60 per cent GTBSA (n= II).
60 1 TBSA lrl=ll)
Figure 3. Maximum plasma endotoxin levels throughout the clinical course in Groups A to C. 0, Survivor; 0, non-survivor; A, non-survivor died within 48 h postinjury.
Bums (1992) Vol. lS/No.
488
Survivors ,”
= 21,
NO” (/i
sYrY,“or*
= 17)
Figure& Maximum levels of plasma endotoxin compared between survivors and non-survivors throughout the clinical course (x, excluding four patients who died within 48 h). 0, Survivor; 0, non-survivor: A, non-survivor died within 48 h postinjury.
significantly higher than in the other two groups. When a comparison was made between the survivors and those with a fatal outcome, plasma endotoxin levels exceeded 9.8 pg/ml in 11/25 (44.0 per cent) of the former group and 13117 (76.5 per cent) of the latter group (Figtrre4). Also, when the four patients who died within 48 h were excluded, all 13 other patients who eventually died developed high endotoxin levels. The plasma endotoxin levels were 44.9 f 124.2 pg/ml in the survivors and 244.9f 278.2 pg/ml in those dying of bums, and the difference was significant, P< 0.05, and P< 0.01 when the four patients dying early were excluded.
Discussion In this study, we used an endotoxin-specific assay (the Endospecy test) that does not react with (I +3)-fi-n-glucan. In addition, pretreatment of plasma by the new PCA method was considered to be important. With the old PCA method for the pretreatment of plasma, SO-90 per cent of the net endotoxin activity was precipitated during processing and discarded, with this discarded endotoxin still having a dose-dependent bioactivity (Imada et al., 1991). When endotoxin levels are measured in bums patients with Endospecy, false-positive reactions are sometimes obtained because of sulpha drug therapy, since such agents undergo diazo-coupling similarly to the nitroaniline produced by degradation of the chromogenic substrate used in the assay. Accordingly, we have developed a method to allow for such false-positive diazo-coupling and thus obtain the net endotoxin levels (Inada et al., 1990), because it is necessary to consider carefully the effect of sulpha drugs in bums patients. Thus, if the endotoxin-specific assay was used with the conventional PCA method for plasma pretreatment, it would still be difficult to assess correctly the relationship of endotoxin levels to the clinical state.
6
It has been reported that major stresses like bums, massive trauma, or haemorrhagic shock produce ischaemia of the intestinal tract, which allows endotoxin and Gramnegative bacteria to enter the blood and gives rise to endotoxaemia (Woodruff et al., 1973; Winchurch et al., 1987; Marek et al., 1989). The translocation of bacteria into the blood from the intestinal tract has been reported (Deitch et al., 1987; Baker et al., 1988) in rats with experimental haemorrhagic shock. However, a recent study on human abdominal trauma suggested that the translocation of intestinal bacteria did not take place (Moore et al., 1991). The present study on human bum injury found that a raised endotoxin level was rare during the week immediately after the accident, including the period of bum shock. It was of interest that in the early period after bum injury, when no infection was present, the endotoxin level did not exceed 9.8 pg/ml, even in patients with bums b 90 per cent BSA. In the early stage after bum injury, there was also no correlation between the extent of the bums and the endotoxin level, unlike the study of Winchurch et al. (1987) which suggested a correlation. In contrast to the theory that the permeability of the intestinal tract membrane increases during the early stage of bum injury and allows the translocation of endotoxin and Gram-negative bacteria (Deitch et al., 1987; O’Dwyer et al., 1988), our results suggested that there was no marked translocation in the first week after the accident. In examining the relationship between endotoxin levels and bum size during the total clinical course, we found that patients with larger percentage bums had a tendency towards increased endotoxin levels. This difference was significant for group C (2 60 per cent burned), when the four patients dying early were excluded. In addition, patients with a fatal outcome had significantly higher endotoxin levels. Since endotoxin levels increased at more than 1 week after the bum injury when infection had become established, it seems possible that infected bums, and not the intestinal tract provided the source of endotoxaemia. Moreover, usually the greater the extent of the bum, the larger the volume of fluid given during resuscitation, however, the endotoxin levels were not related to the volumes of fluid given in the early period after bum injury. We conclude that endotoxaemia in bums patients was possibly caused by endotoxin or Gram-negative bacteria from infected lesions rather than translocating from the intestinal tract.
Acknowledgements This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan, and from the Marumo Critical Care Medicine Research Foundation.
References Baker J. W., Deitch E. D., Berg R. D. et al. (1988) Hemorrhagic shock induces bacterial translocation from the gut.]. Trauma 28, 896. Deitch E. A. and Rodney D. A. (1987) Endotoxin but not malnutrition promotes bacterial translocation of the gut flora in burned mice. 1. Trauma 27, 161. &itch
E. A., Berg R. and Specian R. (1987) Endotoxin promotes the translocation of bacteria from the gut. Arch. Surg. 122,185.
Endo et al.: Plasma endotoxin levels in burned patients
Horn J. K., Goldstein 1. M. and Flick M. R. (1984) Complement and endotoxin-induced lung injury in sheep. 1, Strrg. Res. 136,420. Inada K., Suzuki M., Endo S. et al. (1990) Measurement of plasma endotoxin levels. ]pn. 1. Bacterial. 45, 937 (in Japanese). Inada K., Endo S., Takahashi K. et al. (1991) Establishment of a new perchloric acid treatment to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application. Mcrobiol. hmunol. 35, 303. Marek K. D., Jan S. D., John L. N. et al. (1989) Endotoxemia after bum injury: effect of early excision on circulating endotoxin levels. 1, Bum Care Rekabil. 10, 107. Mason A. D., McManus A. T. and Pruitt B. A. (1986) Association of burn mortality and bacteria. Arch. Surg. 121,1027. Moore F. A., Moore E. E., Poggetto R. et al. (1991) Gut bacterial translocation via the portal vein: a clinical perspective with major torso trauma. 1. Trauma 31, 629. Obayashi T., Tamura H., Tanaka S. et al. (1985) New chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical application. Clin. Ckim. Acta 149, 55.
489
O’Dwyer S. T., Michie H. R., Ziegler T. R. et al. (1988) A single dose of endotoxin increases intestinal permeability in healthy humans. Arch. Surg. 123, 1459. Takahashi K. (1988) Study on quantitative measurement of endotoxin in human blood using chromogenic substrate. Especially pretreatment of plasma. 1, hate Med. Assoc. 40,67 (in Japanese). Winchurch R. A,, Thupari J. N. and Munster A. M. (1987) Endotoxemia in bum patients: levels of circulating endotoxins are related to bum size. Surgery 102, 808. Woodruff I’. W. H., O’Carroll D. I., Koizumi S. et al. (1973) Role of intestinal flora in major trauma. 1. Infect. Dis. 128,290. Paper accepted
31 July 1992.
Correspondence should be addressed to: Dr Shigeatsu Endo, Critical Care and Emergency Center, Iwate Medical University, 19-1 Uchimaru, Morioka 020, Japan,
486
Printed in Great Britain
Are plasma endotoxin levels related to burn size and prognosis? S. Endo’, K. Inadaz, M. Kikuchi’, Y. Yamadal, T. Kasoi’, M. Kondo*, S. Hoshi’, H. Yamashitaz, M. Suzuki2 and M. Yoshida’ ‘Critical Care and Emergency Morioka, Japan
Center and ‘Department
of Bacteriology,
Plasma endofoxin concentrations were determined in 42 pafienfs with burns covering more than 20 per cent of the body surface area, using the endofoxin-specific Endospecy assay and freafmenf of plasma by a new method developed by ourselves. The normal endofoxin level was 9.8pglml or less. In the early period affer injury when no infection was present, very few patients had an endofoxin level above 9.8pglml and endofoxin levels did not correlate with the area of fhe bums or wifh prognosis. However, later in the clinical course, endofoxin levels were correlated sigr#icunfly with the burned area and with the prognosis.
Introduction Along with recent progress in both medical and surgical management, the results achieved in the treatment of bums have been improved markedly. However, infection still remains a major problem during bums treatment. It is thought that endotoxin derived from Gram-negative bacteria has a role in various complications which burns patients develop (Horn et al., 1984; Mason et al., 1986). There are reports of endotoxin entering the blood from the intestinal tract in the early stages of bum injury, and as the burned area becomes larger the blood endotoxin level apparently increases (Woodruff et al., 1973; Deitch and Rodney, 1987; Winchurch et al., 1987; Marek et al., 1989). The conventional assay methods were not specific for endotoxin, but an endotoxin-specific assay (Endospecy) was developed several years ago (Obayashi et al., 1985). Moreover, it was almost impossible to measure accurately the plasma endotoxin level after pretreatment of plasma samples by the conventional perchloric acid method, so we developed a new method for the pretreatment of plasma (Takahashi, 1988; Inada et al., 1991). This new pretreatment method made it possible to obtain fairly accurate plasma endotoxin levels using the Endospecy test. In this study, we monitored plasma endotoxin levels in bums patients from the time of admission and investigated the relationship of endotoxin to bum size and clinical outcome.
Materials and methods Patients and blood collection The subjects were 42 patients with bums affecting not less than 20 per cent of their body surface area (BSA) who could 0 1992 Butterworth-Heineman 030554179/92/060486-04
Ltd
School of Medicine,
Iwate Medical University,
undergo successive blood tests from early after their admission. They included 24 males and 18 females aged from 7 to 87 years (mean f s.d., 47.5 f 23.7 years). The bum size ranged from 20 to 95 per cent BSA (45.5 f 21.5 per cent). The subjects were divided into three groups according to the burned area: group A, 20-39 per cent BSA (26.8 f 6.3 per cent, n = 18); group B, 40-59 per cent BSA (47.4 f 4.7 per cent, n = 13); and group C 2 60 per cent BSA (76.8 f 13.2 per cent, n = 11). There were 25 survivors and 17 patients died. In all patients, blood collection was started within 2 h after injury. Blood samples were taken every 4 h in the first 24 h after admission, every 8 h in the period from 24 to 72 h, and every 12 h in the period from 72 h to 1 week. After 1 week, blood was taken once a day, unless disseminated intravascular coagulation (DIC) or septic shock was observed, in which case it was taken every 2-3 h. Blood collection was continued until death or until no risk of infection remained. Blood was collected into heparinized tubes and centrifuged at 3000 r.p.m. for 40s to obtain plasma, which was stored at - 80°C until assayed. Endotoxin assay The plasma endotoxin level was measured using an endotoxin-specific chromogenic limulus test, the Endospecy test (Seikagaku Corporation, Tokyo, Japan). Plasma was pretreated by the new perchloric acid method (new PCA method) to remove factors interfering with the limulus test. The accuracy of measurement of endotoxin after the new PCA pretreatment method was improved about eight-fold (Takahashi, 1988; Inada et al., 1991) when compared to conventional PCA pretreatment of plasma (Obayashi et al., 1985). When using the conventional PCA method, the precipitate formed after PCA addition is discarded and the supematant is assayed for endotoxin. However, most of the plasma endotoxin activity is actually contained in the discarded precipitate (Inada et al., 1990). Accordingly, we devised a new PCA treatment method which allowed this endotoxin to be retained. Brieftly, 100 pl of 0.18 N NaOH was added to a 100 pl sample of test plasma and incubated at 37”C, 5 min. Then 100 ~1 of 0.32 M PCA was added and further incubation was performed at 3 7°C for 10 min. The precipitate was then dissolved in 200 pl of 0.18 N NaOH by vigorous mixing with a vortex mixer. Finally, 50 pl of the
Endo et al.: Plasma endotoxin levels in burned patients
487
solution was transferred to another tube, and 50 ~1 of 0.2 M Tris-HCl (pH 8.0) was added. Thus, the plasma sample was diluted tenfold. An aliquot (100 ~1) of this diluted plasma was then used for the Endospecy limulus test. This test amoebocyte lysate obtained from the utilizes Japanese horseshoe crab, Tachypletls tridents&. For quantitative measurement, a synthetic chromogenic substrate (n-tertbutoxycarbonyl-L-leucy-L-glycyl-L-argininep-nitroaniline) was added to the lysate to act as the substrate for the clotting enzyme. A 100 ~1 aliquot of Endospecy was added to 100 ~1 of treated plasma, mixed for several seconds and then incubated at 37°C for 30 min. The amount of P-nitroaniline (pNA) released from the substrate was detected after diazo-coupling. The absorbance of the solution was measured at 545 nm, and a standard curve was plotted on a bilogarithmic scale using lipopolysaccaride solution (E. cd 0111:B4, Difco). The normal endotoxin level as determined by this method was less than 9.8 pg/ml (Inada et al., 1991). With the Endospecy test, false-positive reactions are sometimes observed in patients receiving sulpha drug therapy. This is due to the presence of an aromatic primary amine in their chemical structure. The false-positive value was obtained by incubating treated samples with water instead of Endospecy, and the true value was then calculated by subtracting the false-positive value from the Endospecy test value. The research protocol was approved by the Institutional Review Board of Iwate Medical University and all the patients gave their informed consent to participation. Statistical
analysis
Data are expressed as the mean & s.d. Statistical analysis was performed with the two-tailed Wilcoxon rank-sum test for unpaired data and the level of significance was set at P< 0.05.
patients remained under 9.8pg/ml in this first week (Figttre I), and no significant difference was observed between them. In addition, no significant difference was noted when plasma endotoxin levels were reviewed on a daily basis. When a comparison was made between the survivors and those who died with respect to plasma endotoxin levels in the first week after admission, no significant difference was observed (F@re 2). Similarly, no significant difference was observed in the daily changes of the plasma endotoxin levels. When the total clinical course was considered, the number of patients with plasma endotoxin levels above 9.8 pg/ml was 5/18 (27.8 per cent) in group A, 11113 (84.6 per cent) in group B and 7/11 (63.3 per cent) in group C (Figure 3). However, if the four patients who died within 48 h were excluded, all seven remaining patients in group C had raised endotoxin levels. The plasma endotoxin levels in groups A, B and C were 44.2 f 142.9, 160.1& 225.2 and 236.6 & 282.5 pg/ml, respectively, and no significant difference was observed among the three groups. However, when the four patients who died early were excluded from group C, the level rose to 391.0 rt 269.8 pg/ml, and this was
5
:: ;; D : E Mean
Results
Time
Only one patient had a plasma endotoxin level above 9.8 pg/ml at the time of admission. In the first week after the bum injury, only five patients (6/956 blood samples, 0.70 per cent) showed plasma endotoxin levels over 9.8pg/ml. The range of elevation was 11.6 pg/ml (10.8 f 0.6 pg/ml).
The mean plasma endotoxin
not
great,
being
postinjury
(days)
V-PC ,--P
o.oL
< 0.05
-NS-
Y-N-Y--~
.
. .Cl
10 2 E _
_________________ _________ ____-- _________
? s.d.
Figure 2. Changes in the plasma endotoxin levels from the time of injury to I week later in survivors (0 -0, n=25) and non-survivors ( l 0, n = 17) (excluding four patients dying within 48 h).
10.2-
levels of all three groups of
.8
10 r-__-----_________________--______---_._.____--_..~------__----~
E -En ,a a, -2 .G
:
.
___ __________________ 9. * .
1
-
P
0
z
Mean
E; 0
0
1
2 Time
/ 3 postinjury
, 5
/ 4
i I 6
s.d. 7
_;_
(days)
Figure 1. Changes in the plasma endotoxin level from the time of injury to I week later (maximum daily levels in relation to bum size as per cent BSA). O0, 20 per cent < TBSA < 40 per cent (n = 18); l 0, 40 per cent d TBSA < 60 per cent (VI= 13); A ---A, 60 per cent GTBSA (n= II).
60 1 TBSA lrl=ll)
Figure 3. Maximum plasma endotoxin levels throughout the clinical course in Groups A to C. 0, Survivor; 0, non-survivor; A, non-survivor died within 48 h postinjury.
Bums (1992) Vol. lS/No.
488
Survivors ,”
= 21,
NO” (/i
sYrY,“or*
= 17)
Figure& Maximum levels of plasma endotoxin compared between survivors and non-survivors throughout the clinical course (x, excluding four patients who died within 48 h). 0, Survivor; 0, non-survivor: A, non-survivor died within 48 h postinjury.
significantly higher than in the other two groups. When a comparison was made between the survivors and those with a fatal outcome, plasma endotoxin levels exceeded 9.8 pg/ml in 11/25 (44.0 per cent) of the former group and 13117 (76.5 per cent) of the latter group (Figtrre4). Also, when the four patients who died within 48 h were excluded, all 13 other patients who eventually died developed high endotoxin levels. The plasma endotoxin levels were 44.9 f 124.2 pg/ml in the survivors and 244.9f 278.2 pg/ml in those dying of bums, and the difference was significant, P< 0.05, and P< 0.01 when the four patients dying early were excluded.
Discussion In this study, we used an endotoxin-specific assay (the Endospecy test) that does not react with (I +3)-fi-n-glucan. In addition, pretreatment of plasma by the new PCA method was considered to be important. With the old PCA method for the pretreatment of plasma, SO-90 per cent of the net endotoxin activity was precipitated during processing and discarded, with this discarded endotoxin still having a dose-dependent bioactivity (Imada et al., 1991). When endotoxin levels are measured in bums patients with Endospecy, false-positive reactions are sometimes obtained because of sulpha drug therapy, since such agents undergo diazo-coupling similarly to the nitroaniline produced by degradation of the chromogenic substrate used in the assay. Accordingly, we have developed a method to allow for such false-positive diazo-coupling and thus obtain the net endotoxin levels (Inada et al., 1990), because it is necessary to consider carefully the effect of sulpha drugs in bums patients. Thus, if the endotoxin-specific assay was used with the conventional PCA method for plasma pretreatment, it would still be difficult to assess correctly the relationship of endotoxin levels to the clinical state.
6
It has been reported that major stresses like bums, massive trauma, or haemorrhagic shock produce ischaemia of the intestinal tract, which allows endotoxin and Gramnegative bacteria to enter the blood and gives rise to endotoxaemia (Woodruff et al., 1973; Winchurch et al., 1987; Marek et al., 1989). The translocation of bacteria into the blood from the intestinal tract has been reported (Deitch et al., 1987; Baker et al., 1988) in rats with experimental haemorrhagic shock. However, a recent study on human abdominal trauma suggested that the translocation of intestinal bacteria did not take place (Moore et al., 1991). The present study on human bum injury found that a raised endotoxin level was rare during the week immediately after the accident, including the period of bum shock. It was of interest that in the early period after bum injury, when no infection was present, the endotoxin level did not exceed 9.8 pg/ml, even in patients with bums b 90 per cent BSA. In the early stage after bum injury, there was also no correlation between the extent of the bums and the endotoxin level, unlike the study of Winchurch et al. (1987) which suggested a correlation. In contrast to the theory that the permeability of the intestinal tract membrane increases during the early stage of bum injury and allows the translocation of endotoxin and Gram-negative bacteria (Deitch et al., 1987; O’Dwyer et al., 1988), our results suggested that there was no marked translocation in the first week after the accident. In examining the relationship between endotoxin levels and bum size during the total clinical course, we found that patients with larger percentage bums had a tendency towards increased endotoxin levels. This difference was significant for group C (2 60 per cent burned), when the four patients dying early were excluded. In addition, patients with a fatal outcome had significantly higher endotoxin levels. Since endotoxin levels increased at more than 1 week after the bum injury when infection had become established, it seems possible that infected bums, and not the intestinal tract provided the source of endotoxaemia. Moreover, usually the greater the extent of the bum, the larger the volume of fluid given during resuscitation, however, the endotoxin levels were not related to the volumes of fluid given in the early period after bum injury. We conclude that endotoxaemia in bums patients was possibly caused by endotoxin or Gram-negative bacteria from infected lesions rather than translocating from the intestinal tract.
Acknowledgements This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan, and from the Marumo Critical Care Medicine Research Foundation.
References Baker J. W., Deitch E. D., Berg R. D. et al. (1988) Hemorrhagic shock induces bacterial translocation from the gut.]. Trauma 28, 896. Deitch E. A. and Rodney D. A. (1987) Endotoxin but not malnutrition promotes bacterial translocation of the gut flora in burned mice. 1. Trauma 27, 161. &itch
E. A., Berg R. and Specian R. (1987) Endotoxin promotes the translocation of bacteria from the gut. Arch. Surg. 122,185.
Endo et al.: Plasma endotoxin levels in burned patients
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31 July 1992.
Correspondence should be addressed to: Dr Shigeatsu Endo, Critical Care and Emergency Center, Iwate Medical University, 19-1 Uchimaru, Morioka 020, Japan,
