Objective　To systemically investigate 1) distribution of endogenous endotoxin (ET) in tissues and circulation; 2) its relationship with shock duration and organ damage; and 3) its possible mechanism after hemorrhagic shock.
　　Methods　To further elucidate the intrinsic relationship between endogenous endotoxin translocation and hemorrhagic shock, the present study systematically investigated the distribution of endogenous ET into the liver, lungs, kidneys and circulation, and the relationship between ET levels and the corresponding organ dysfunction with limulus amebocyte lysate (LAL) chromogenic assay following hemorrhagic shock in rats.
　　Results　It was found that ET levels in hepatic homogenate markedly increased (P=0.09) 1.5 hours following shock compared with that in the sham group. After resuscitation, ET levels in hepatic, pulmonary and renal tissues were all significantly elevated. The levels kept increasing with the prolonged experimental time, and reached as high as 3.88±0.95 EU (endotoxin unit)/g in the livers, 2.53±1.46 EU/g in the lungs and 2.51±0.89 EU/g in the kidneys 12 hours after shock. ET levels in plasma reached a peak of 1.13±0.42 EU/ml at 1 hour following resuscitation, then rapidly decreased to the sham levels 3 hours following resuscitation. There was a close relationship between endotoxin translocation and shock duration. Correlation analysis further indicated that the changes in glutamic-pyruvic transaminase (GPT), blood urea nitrogen (BUN) in plasma and angiotensin Ⅰ-converting exzyme (ACE) in pulmonary homogenate were significantly and positively correlated with the ET levels in the liver, kidneys and lungs after hemorrhagic shock.
　　Conclusions　Hemorrhagic shock can induce obvious endogenous ET translocation, which is closely related to the shock duration. Although only transient endotoxemia occurs after hemorrhagic shock, ET can massively accumulate in tissues (liver, lungs and kidneys), and may play an important role in the development of shock.

　　Although the close relationship of hemorrhagic shock and endotoxemia has been demonstrated by many investigators,1-3　the role of endotoxemia in the development of shock is still obscure4-6　because of transient and weak elevation of plasma endotoxin (ET) following hemorrhagic shock and subsequent resuscitation. It has been recently found that majority of lipopolysaccharide (LPS) could rapidly enter the tissues from circulation within several minutes after intravenous injection of LPS,7-9　and keep its toxicity for a long time,9 suggesting that plasma ET levels can not accurately reflect the in vivo bioactivities of ET, and that ET in tissues may be more directly responsible for organ dysfunction after shock. However, there is little information concerning the quantitative changes of ET levels in tissues and its relationship with the corresponding organ dysfunction following shock.

　　The purpose of the present study is to explore the kinetics of endogenous ET in the liver, lungs, kidneys and circulation, investigate the relationship between ET translocation and shock duration, and the correlation of ET levels in tissues with the corresponding organ damages, in order to further elucidate the intrinsic relationship between endogenous ET translocation and hemorrhagic shock.

METHODS

Animals and models
　　Wistar rats in both sexes, weighing 170±13 g, were used in this experiment. They were fed with standard laboratory chew and water. The animals were fasted overnight with water and libitum prior to the experiment.

　　After anesthetization with 3% of sodium pentobarbital (35 mg/kg, i.p.), the animals were fixed onto a surgical board in the supine position. By applying aseptic technique, a carotid artery was cannulated with a polyethylene catheter which was then connected to a pressure transducer and a two-channel physiological recorder via a three-way stopcock to determine the mean arterial blood pressure (MAP) and also connected to a syringe to monitor bleeding. The animals were heparinized ［250 IU　(international unit) /kg, i.a.］. The materials used in this experiment were sterile and pyrogen-free.

　　The animals were bled to MAP of 4.00—4.65 kPa (30-35 mmHg; 1 kPa=7.5 mmHg) over 10 minutes and maintained at the level for 90 minutes. The shed blood was then returned followed by the infusion of Ringer's lactate, equivalent to the shed blood volume (about 30 minutes). The sham animals underwent the same surgical procedure but without hemorrhage.

Endotoxin distribution in tissues following hemorrhagic shock
　　Seventy-nine rats were used and randomly divided into shock (n=40) and sham (n=39) groups. The animals were respectively sacrificed 1.5 hours following shock, and 0, 1, 3 and 10 hours after resuscitation (n=8, per time point; but n=7 for 1.5 hour time point in sham group). The samples of the blood, liver, lungs and kidneys were taken at each time point. Plasma were immediately separated and stored at -30℃, and tissue samples were stored in liquid nitrogen.

Relationship between endotoxin translocation and shock duration
　　Thirty-two rats were used and randomly divided into 0.5-, 1-, 1.5- and 2-hour shock groups (n=8). MAP of 4-4.65 kPa was maintained for 0.5, 1, 1.5 and 2 hours. Immediately after resuscitation, the animals were sacrificed. Samples of blood and tissues were taken out.

Assay of ET
　　Limulus amebocyte lysate test was used.10　All materials used in this experiment before the terminal reaction were sterile and pyrogen-free. The tissue samples were weighed and homogenized in pyrogen-free physiological saline (1∶10, weight/volume). The homogenates were then heated at 100℃ for 10 minutes. After centrifugalization, the supernatant was taken out for ET assay. The plasma samples were diluted with pyrogen-free water in a ratio of 1∶10 and then heated at 100℃ for 5 minutes to remove non-specific inhibitors before assay. ET concentrations in EU/g or EU/ml were calculated from the optical densities, according to a standard curve of E. coli B26∶06 endotoxin (Difco) and recovery rate.

Assay of pulmonary angiotensin Ⅰ-converting enzyme
　　Lung tissue, weighing 0.1 g, was taken out from the liquid nitrogen, and then minced in liquid nitrogen. 1 ml of 0.01 mol/L Tris-HCl buffer (pH 7.4) was added and homogenized with tissue homogenizer. Angiotensin I-converting enzyme (ACE) contents were assayed with a ACE test kit (Navy General Hospital). Plasma glutamic-pyruvic transaminase (GPT) and blood urea nitrogen (BUN) levels were assayed by spectrophotometry.

Statistics
　　Data were presented as ±s. Student's t test and correlation analysis were used for statistical test. P<0.05 was considered significant, and P<0.01 very significant.

RESULTS

Development of assay of ET in tissue homogenate
Linear relationship between amount of LPS and its absorption
　　As shown in Fig. 1, there was a good linear relationship between the amount of standard LPS in tissue homogenate and its absorption (A) value (γ=0.953).
 

 

Fig. 1. Linear relationship between LPS contents in liver homogenate and absorption values (γ=0.953).
Precision
　　Three samples were simultaneously assayed with double tubes. The intra-assay precision was 3.8%. The same sample was assayed 5 times within successive 5 days. The inter-assay precision was shown to be 11%.

Recovery
　　It was shown to be 80.5%, 78.5% and 80.5% for the liver, lungs and kidneys, respectively.

Sensitivity
　　The mean absorption value was 0.124 for 0.1 EU/ml of LPS in liver homogenate, which was significantly different from that of blank control (A=0.098), suggesting that the sensitivity is 0.1 EU/ml (about 10 pg/ml).

Distribution of endogenous ET into tissues and circulation and its effects on the corresponding organ function following hemorrhagic shock
The shed volume and blood pressure
　　The final shed volume to keep MAP of 4.00—4.67 kPa for 1.5 hours in rats was 6±1.2 ml, accounting for 59% of the circulating volume (calculating as 6 ml per 100 gram of body weight11). Changes of MAP are shown in Fig. 2.
 

 

Fig. 2. Changes of mean arterial blood pressure (MAP) following hemorrhagic shock and resuscitation. B: baseline values.
ET levels in tissues and plasma
　　One and a half hours following shock, ET levels in liver tissue began to be markedly increased compared with those in the sham group (P=0.09). At the end of resuscitation, ET levels in the hepatic, pulmonary and renal tissues and in the plasma were all significantly increased compared with those in the sham group (P<0.05 and P<0.01). ET levels in tissues kept increasing with the prolonged experimental time, and 10 hours after resuscitation, the levels reached 3.88±0.95 EU/g in the liver, 2.53±1.46 EU/g in the lungs and 2.51±0.89 EU/g in the kidneys; while in plasma, ET levels reached a peak of 1.13±0.42 EU/ml 1 hour after resuscitation, then rapidly decreased to the sham values 3 hours after resuscitation. ET levels in the liver were significantly higher than those in the lungs and kidneys through the experiment (Fig.3).
 

 

Fig. 3. Kinetics of endotoxin (ET) levels in tissues and plasma following hemorrhagic shock. ES: end of shock. * P<0.05, ** P<0.01, vs the sham group.
GPT, BUN levels in plasma and ACE levels in pulmonary homogenate
　　As shown in Fig.4, GPT and BUN levels in plasma and ACE levels in pulmonary homogenate were obviously elevated after shock and subsequent resuscitation.

Correlation analysis
　　GPT and BUN levels in plasma and ACE levels in pulmonary homogenate were significantly and positively correlated with ET levels in the corresponding tissues (Table 1.).

Table 1. Correlation analysis of endotoxin levels
in tissues with organ damages
 

　 Correlation coefficient (γ) P values
Liver Lung Kidney
GPT 0.99 　 　 0.01
ACE 　 0.964 　 0.01
BUN 　 　 0.949 0.05

　　GPT: plasma glutamic-pyruvic transaminase; BUN: blood urea
nitrogen; ACE: angiotensin Ⅰ-converting enzyme.

 

Fig. 4. Changes of plasma glutamic-pyruvic transaminase (GPT), blood urea nitrogen (BUN) and pulmonary angiotensin Ⅰ-converting exzyme (ACE) levels following hemorrhagic shock. ES: end of shock. # P<0.05, * P<0.01, compared with the sham group.
Table 2. Relationship between endotoxin levels in plasma and
tissues and shock duration
 

　 Shock duration
0.5 h 1.0 h 1.5 h 2.0 h
Plasma 0.32±0.16 0.68±0.30 1.06±0.47☆ 1.47±0.45★▲ 
Liver 0.75±0.25 1.67±0.74★ 1.94±0.84★ 3.94±1.25★▲
Lung 0.28±0.10 0.47±0.21 1.00±0.45★ 2.51±0.90★▲
Kidney 0.20±0.09 0.66±0.29☆ 0.70±0.45☆ 1.25±0.48★△

　　☆ P<0.05, ★ P<0.01, compared with the 0.5 h group;
△ P<0.05, ▲ P<0.01, compared with the 1.0 h group.
Relationship between endogenous ET translocation and duration of hemorrhagic shock
　　As shown in Table 2, the longer the shock duration,the higher the ET levels in tissues and plasma.

DISCUSSION

　　Although there is more and more evidence showing that transient and weak endotoxemia often occurs at the early stage of hemorrhagic shock,1-3　antagonists against endotoxin (antiserum to ET, lactulose) are shown to have obvious protection effect against hemorrhagic shock.12-14　It is hard to explain the close intrinsic relationship between ET translocation occurring after hemorrhagic shock and the outcome of shock just according to plasma ET levels after shock. We hypothesized that ET in tissues might be directly related to sepsis and multiple organ dysfunction syndrome (MODS) which occur after shock.

　　Based on the modification of LAL chromogenic test, we developed an assay of ET in tissue homogenate. The results showed that (1) there was a good linear relationship between ET contents in tissue homogenate and its absorption value (γ=0.953), suggesting that the factors which interfere LAL assay could be basically removed by means of homogenizing tissue samples with physiologic saline and heat treatment; (2) both of intra- and inter-assay precision coefficients were 3.8% and 11%, suggesting that the assay has a good accuracy; (3) the recovery rate was all shown to be about 80% in the liver, lungs and kidneys, suggesting that only small amount of ET was lost during the preparation of tissue samples; (4) the sensitivity (the smallest amount that could be measured reliably) was 0.1 EU/ml (about 10 picogram/ml). The above results indicate that LAL assay can be also used for quantitative assessment of ET contents in tissue homogenate, for its good sensitivity, reproduction, and recovery. By using LAL assay of tissue and plasma ET, it was found in this experiment that ET levels in hepatic homogenate began to be markedly increased 1.5 h following shock compared with those in the sham group (P=0.09). ET contents in the liver, lungs and kidneys were all significantly elevated after resuscitation, and kept increasing with the prolonged experimental time, reached 3.88±0.95 EU/g in the liver, 2.53±1.46 EU/g in the lungs and 2.51±0.89 EU/g in the kidneys 10 hours after resuscitation. However, ET levels in plasma reached a peak of 1.13±0.42 EU/ml 1 hour after resuscitation, then rapidly decreased to the sham levels 3 hours after resuscitation. The results indicate that although only transient endotoxemia occurs after hemorrhagic shock, ET can be massively distributed in tissues (the liver, lungs and kidneys). The present experiment further indicates that there is a close relationship between endotoxin translocation and duration of shock, that is, the longer the duration of shock, the higher the ET levels in tissues and plasma. The present results also showed that ET levels in the liver, lungs and kidneys were not decreased, but even increased 12 hours following shock when compared with those in the post-shock 5 hour group, which were obviously different from the changes of ET levels in plasma, suggesting that ET can keep its bioactivity in tissues for long time. Ayala et al15　have reported that a slight decrease in blood pressure (6.67 kPa for 1 hour) or temporary shock (4.65 kPa for 15 minutes) was enough to induce suppression of antigen presentation of monocytes/macrophages for as long as 120 hours. Phagocytosis of monocyte/macrophage system was also decreased by over 50% 3 hours after hemorrhagic shock.16　Detoxification of ET in vivo mainly depends upon deacylation and dephosphorylation by phagocytes. Therefore, it is concluded that suppression of monocyte/macrophage system following hemorrhagic shock might be responsible for the prolonged existence of ET in tissues after shock.

　　It was further shown in the present study that ET levels in hepatic homogenate were firstly increased 1.5 hours following shock, and were higher than those in pulmonary and renal homogenates throughout the experiment. ET levels in pulmonary homogenate were also higher than those in renal tissue following shock and subsequent resuscitation. It is suggested that there are differences in ET distribution among different tissues after hemorrhagic shock. The liver is the most important organ for ET accumulation after hemorrhagic shock, for 1) the portal circulation is the prominent route for ET in the intestine to enter the body after hemorrhagic shock;17　and 2) the liver is the largest organ of monocyte/macrophage system, from where ET is mainly eliminated out of the body. According to the above results, ET translocated into the body after hemorrhagic shock was mainly distributed in the tissue organs and remained its bioactivities in tissues for a long time. To what extent ET in tissues is responsible for organ dysfunction and outcome of shock? Our experiment showed that changes of GPT and BUN levels in plasma and ACE levels in pulmonary homogenate were significantly correlated with the changes of ET in the corresponding tissues, suggesting that there is a certain intrinsic relationship between accumulation of ET in tissues and organ dysfunction following hemorrhagic shock. Furthermore, it was recently reported that antagonism of ET effects, such as anti-LPS antibody,13,14　polymyxin B,18　and bactericidal/permeability-increasing protein (BPI),19　could obviously ameliorate organ damage, inhibit production of cytokines and improve survival rate of the shocked animals after hemorrhagic shock. On the basis of the above analysis, it is concluded that although there is transient and weak endotoxemia occurring at the early stage of hemorrhagic shock, ET translocation still plays an important role in the development of shock.

　　In conclusion, the present study, for the first time, systematically examined the kinetics of endogenous ET in tissue organs and investigated its relationship with organ damage in an animal model of hemorrhagic shock. It is found that hemorrhagic shock can cause remarkable ET translocation. Although only transient endotoxemia occurs after hemorrhagic shock, the translocated ET following shock can massively accumulate in tissues, such as the liver, lungs and kidneys. Endotoxin translocation is closely related to the duration of shock. Endotoxin translocation might play an important role in the development of shock, which may be mainly related to the accumulation of endotoxin in tissues.

Research Institute of Surgery, The Third Military Medical College, Chongqing 400042, China (Jiang JX, Chen HS, Diao YF, Tian KL, Zhu PF and Wang ZG)

REFERENCES

　　1. Jiang J, Bahrami S, Leitrifter G, et al. Kinetics of endotoxin and TNF appearance in portal and systemic circulation after hemorrhagic shock in rats. Ann Surg 1995; 221(1):100.
　　2. Bottoms G, Gimarc S, Pfeifer C. Plasma concentration of endotoxin following jugular injections of endotoxin and following gastrointestinal ischemia due to hemorrhage. Circ Shock 1991; 33:1.
　　3. Rush BF, Sori AJ, Murphy TF, et al. Endotoxemia and bacteremia during hemorrhagic shock. Ann Surg 1988; 207(5):549.
　　4. Schmand JF, Ayala A, Chaudry IH. Effects of trauma, duration of hypotension, and resuscitation regimen on cellular immunity after hemorrhagic shock. Crit Care Med 1994; 22:1076.
　　5. Endo S, Inada K, Yamada Y, et al. Plasma endotoxin and cytokine concentrations in patients with hemorrhagic shock. Crit Care Med 1994; 22:949.
　　6. Munster AM, Smith-Meek M, Dickerson C, et al. Translocation: incidental phenomenon or true pathology? Ann Surg 1993; 218:321.
　　7. Nakao A, Shimonhara M. Changes of circulating blood endotoxin analyzed by quantitative assay after intravenous administration of endotoxin. Jpn J Gastroenterol 1985; 82;296.
　　8. Freudenberg N, Freudenberg MA, Bandara K, et al. Distribution and localization of endotoxin in the reticuloendothelial system (RES) and in main vessels of the rat during shock. Path Res Pract 1985; 179:517.
　　9. Van Bossuyt H, De Zanger RB, Wisse E. Cellular and subcellular distribution of injected lipopolysaccharide in rat liver and its inactivation by bile salts. J Hepatol 1988; 7:325.
　　10. Jiang JX, Chen HS, Diao YF, et al. Quantitative assay of low-level endotoxin in tissue homogenates with limulus amebocyte lysate chromogenic assay and its application. Acad J Third Milit Med Univers 1997; 19:S40.
　　11. Fox JG, Cohen BJ, Loew FM. Laboratory animal medicine. New York: American College of Laboratory Animal Medicine Series, 1990:80-98.
　　12. Demaria EJ, Pellicane JV, Lee RB. Hemorrhagic shock in endotoxin-resistant mice: improved survival unrelated to deficient production of tumor necrosis factor. J Trauma 1993; 35:720.
　　13. Gaffin SL, Grinberg Z, Abraham C, et al. Protection against hemorrhagic shock in the cat by human plasma containing endotoxin-specific antibodies. J Surg Res 1981; 31:18.
　　14. Pohlson EC, Suehiro A, Ziegler EJ, et al. Antiserum to endotoxin in hemorrhagic shock. J Surg Res 1988; 45:467.
　　15. Ayala A, Perrin MM, Wagner MA, et al. Enhanced susceptibility to sepsis following simple hemorrhage: depression of Fc and C3b receptor-mediated phagocytosis. Arch Surg 1990; 125:70.
　　16. Zweibach BW, Benacerraf BJ. Effect of hemorrhagic shock on the phagocytic function of kupffer cells. Circ Res 1958; 6:83.
　　17. Olofsson P, Nylander G, Olsson P. Endotoxin-transport routes and kinetics in intestinal ischemia. Acta Chir Scand 1985; 151:635.
　　18. Tian HM, Yao YM, Wang YP, et al. Experimental study on gut-derived endotoxemia and the changes of TNF, IL-1 levels following hemorrhagic shock. Circ Shock 1993; Suppl 1:11.
　　19. Yao YM, Bahrami S, Leichtfried G, et al. Pathogenesis of hemorrhage-induced bacteria/endotoxin translocation in rats: effects of recombinant bactericidal/permeability-increasing protein. Ann Surg 1995; 221:398.