Dieter Berger

Endotoxemia and acute-phase proteins in major abdominal surgery.

BACKGROUND Translocation of endotoxin is a controversial issue. The ability of plasma to inactivate endotoxin is an indirect measure of endotoxemia. Endotoxin is a potent stimulator of the inflammatory response and affects the innate immune system. OBJECTIVE To elucidate the kinetics of endotoxemia and the ability of plasma to inactivate endotoxin in patients with major abdominal operations. To demonstrate the early time course of the acute-phase proteins C-reactive protein (CRP), serum amyloid A (SAA), alpha(1)-antitrypsin, alpha(2)-macroglobulin, transferrin, and interleukin 6 (IL-6), and to correlate them with the amount of endotoxemia. METHODS Twenty patients with elective major abdominal operation and 10 healthy controls were investigated. Blood was collected preoperatively, during the operation and regularly up to 12 days after surgery. Endotoxin was measured by Limulus amebocyte lysate test (LAL), the ability of plasma to inactivate endotoxin by modified LAL, the acute-phase proteins nephelometrically, and IL-6 by enzyme-linked immunosorbent assay (ELISA). RESULTS Preoperative endotoxin plasma level (0.026 +/- 0.004 EU/mL) did not differ from healthy volunteers but increased during operation (0.09 +/- 0.02 EU/mL, P = 0.02). Endotoxemia peaked 1 hour after the surgical procedure (0.16 +/- 0.03 EU/mL; P <0.0001 versus preoperative) and decreased to almost normal values after 48 hours. The capability of plasma to inactivate endotoxin was significantly reduced during (recovery, 0.16 +/- 0.03 EU/mL), 1 hour (0.25 +/- 0.04 EU/mL) and 24 hours (0.16 +/- 0.02 EU/mL) after the operation compared with preoperative (0.068 +/- 0.01 EU/mL) values. Plasma IL-6 was significantly increased for 48 hours with a peak 1 hour after surgery (470 +/- 108 pg/mL). CRP peaked at 210 +/- 19 mg/L (P <0.0001 versus preoperative) 48 hours after operation and was significantly elevated for the rest of the observation period. SAA was significantly increased 24 hours after surgery (249 +/- 45 mg/L) and peaked additional 48 hours later (456 +/- 86 mg/L). alpha(1)-Antitrypsin, although a positive acute-phase protein, decreased initially to 1.38 +/- 0.1 g/L (preoperative, 2.33 +/- 0.18 g/L; P <0.0001) and increased thereafter until day 12 (3.05 +/- 0.35 g/L, P = 0.11 versus preoperative). The same was true for alpha(2)-macroglobulin (preoperative, 2.2 +/- 0.16 g/L; intraoperative, 1.36 +/- 0.13 g/L; day 5, 2.8 +/- 0.4 g/L). Transferrin decreased already during surgery (1.6 +/- 0.1 g/L versus preoperative 2.8 +/- 0.17 g/L, P <0.0001) and remained on this level for 5 days. Correlation analysis revealed a relationship between endotoxemia and the ability of plasma to inactivate endotoxin (r = 0.67, P <0.0001) and also a relation between intraoperative endotoxemia on one hand and alpha(2)-macroglobulin (-0.53 > r > -0.6, P <0.05) as well as alpha(1)-antitrypsin (0.64 > r >0.55, P <0.05) on the other. CONCLUSION Major abdominal surgery is associated with transient endotoxemia and a transient reduced endotoxin inactivation capacity of the plasma. Endotoxemia correlates with the endotoxin inactivation capacity. The surgical procedure causes substantial changes in plasma concentrations of acute-phase proteins. alpha(2)-Macroglobulin and alpha(1)-antitrypsin correlate moderately with endotoxemia.

Preoperative Immunonutrition Suppresses Perioperative Inflammatory Response in Patients with Major Abdominal Surgery—A Randomized Controlled Pilot Study

Background/AimPerioperative administration of immunoenriched diets attenuates the perioperative inflammatory response and reduces postoperative infection complications. However, many questions still remain unresolved in this area, such as the length of diet administration, diet composition, and the mechanisms involved. We performed an open, randomized, triple-arm study comparing the effect of two perioperative feeding regimens with a postoperative one.Methods46 candidates for major elective surgery for malignancy in the upper gastrointestinal tract were randomized to drink preoperatively either 1 L of an immunoenriched formula (Impact) for 5 days (IEF group) or 1 L of Impact plus (Impact enriched with glycine) for 2 days (IEF plus group). The same product as the patient received preoperatively was given to both groups for 7 days postoperatively. In the control group (CON group), patients only received Impact for 7 days postoperatively; there was no preoperative treatment. The main outcome measures were postoperative C-reactive protein (CRP) serum levels.ResultsIn the two preoperatively supplemented groups (treatment groups), perioperative endotoxin levels, CRP (postoperative day 7), and TNF-α (postoperative days 1 and 3) levels were significantly lower compared to the CON group (p < .01). Furthermore, the length of postoperative IMU/ICU stay (Impact 1.9 ± 1.3 days; Impact plus 2.2 ± 1.1 days; control group 5.9 ± 0.8 days) and length of hospital stay (Impact 19.7 ± 2.3 days; Impact plus 20.1 ± 1.3 days; control group 29.1 ± 3.6 days) were both reduced in the treatment groups compared to the control group. Infectious complications (Impact 2/14 (14%); Impact plus 5/17 (29%); control group 10/15 (67%)) also showed a trend toward reduction in the treatment groups.ConclusionsPerioperative administration of an immunoenriched diet significantly reduces systemic perioperative inflammation and postoperative complications in patients undergoing major abdominal cancer surgery, when compared with postoperative diet administration alone. A shortened preoperative feeding regimen of 2 days with Impact enriched with glycine (Impact plus) was as effective as Impact administered for 5 days preoperatively.

Evidence for endotoxin binding capacity of human Gc-globulin and transferrin

In the present paper the ability of Gc-globulin and transferrin to bind endotoxin of Escherichia coli 0 111: B 4 is demonstrated. This conclusion is based on four lines of evidence. By affinity chromatography using lipopolysaccharide of E. coli 0 111: B 4 two endotoxin-binding proteins of serum were identified, showing an apparent molecular weight of 77,000 and 51,000, respectively. If serum samples preincubated with the tritiated endotoxin form have undergone isoelectric focusing under non-denaturing conditions one radioactive peak appears which coincides with the precipitate obtained by immunoelectrophoresis against anti-human Gc-globulin and anti-human transferrin. Radioimmunoprecipitation experiments of serum showed that tritiated endotoxin of E. coli 0 111: B 4 was only found in the precipitate obtained with anti-Gc-globulin, antitransferrin, and polyvalent antiserum against human serum. By isoelectric focusing of purified proteins 3H-lipopolysaccharide of E. coli 0 111: B 4 was only found associated with human Gc-globulin and transferrin.

Endotoxemia and specific antibody behavior against different endotoxins following multiple injuries

The aim of this study was to establish the incidence of endotoxemia and the influence of endotoxin on specific antibody response after multiple injury. Blood samples were collected from 39 patients (median Injury Severity Score: 20.5) at 0-3 and 6-12 hours, and 1, 3, 5, and 10 days after admission. The endotoxin plasma levels were high at the first time point (mean = 0.421 endotoxin units/mL) and decreased in the later course. Total immunoglobulin levels of IgM, IgG, or IgA were low and increased throughout the observation period. Specific antibodies of the IgM class against two lipid A and four lipopolysaccharide preparations increased transiently but significantly on day 3 and/or day 5. No changes of specific antibody content against endotoxin or lipid A was seen in the IgG or IgA class. The specific antibody content of the different classes against alpha-hemolysin of Staphylococcus aureus did not differ during 10 days after trauma. The specific antibodies of the IgM class reacted with all lipid A and LPS lipopolysaccharide preparations demonstrating cross-reactivity. These results suggest that endotoxin may be a specific stimulator of IgM antiendotoxin antibody secretion following trauma.

Time-scale of interleukin-6, myeloid related proteins (MRP), C reactive protein (CRP), and endotoxin plasma levels during the postoperative acute phase reaction.

During goitre surgery (25 patients) and after major abdominal surgery (52 patients), we studied the plasma levels of endotoxin, interleukin-6 (IL-6), C reactive protein (CRP), and the so called myeloid-related proteins (MRP), MRP8, MRP14, and the heterocomplex of both single proteins, MRP8/MRP14 in three intervals: pre-, intra-, and postoperative. We observed that CRP levels began to increase on the first postoperative day, reaching a maximum on day 2 (median levels of 185 mg/L after major surgery and 77 mg/L after goitre surgery). IL-6 levels peaked at the end of the operation, remaining elevated for 6 h following abdominal surgery (299 pg/mL) and peaked on day 1 after goitre surgery (63 pg/mL). An increase in MRP8/MRP14 levels began toward the end of abdominal surgery, and maximum levels were recorded until 5 days after the operation (5,695 μg/L). Plasma levels were significantly elevated 2 and 6 h after minor surgery (3,619 μg/L), while no changes were observed in the plasma levels of MRP8 and MRP14. Evidence of significant endotoxemia was found after the induction of anesthesia in the abdominal surgery group (.13 endotoxin units (EU)/mL) and after skin incision (.07 EU/mL) in the thyroid surgery group. The observed time sequence, starting with the release of bacterial products at an early stage, followed by the secondary stimulation of factors inherent to the acute phase led us to conclude that certain bacterial compounds, probably deriving from the gastrointestinal tract, trigger the postoperative acute phase reaction and are responsible for the activation of monocytes/macrophages and granulocytes.

New aspects concerning the regulation of the postoperative acute phase reaction during cardiac surgery

During a cardio-pulmonary bypass, as well as post-operatively, high levels of endotoxin, interleukin-6 (Il-6) and C-reactive protein (CRP) were measured in 30 patients. A significant increase in endotoxin plasma level occurred during surgery, culminating in a peak during reperfusion. Plasma levels of endotoxin continued to be slightly raised until the fifth day after surgery, whereas those of Il-6 rose at the time the operation came to an end and were at their highest 6 h later. CRP levels were also high, post-operatively, and were markedly raised on day 2. A definite, statistically significant correlation between the plasma levels of endotoxin and Il-6 during the operation was established, leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the release of Il-6 and thus induces the post-operative acute phase reaction. There was no evidence of a correlation between CRP and endotoxin or Il-6 plasma levels.

Endotoxin and antiendotoxin antibodies in patients with acute pancreatitis

OBJECTIVE To elucidate the time course of endotoxaemia and antiendotoxin antibodies in patients with acute pancreatitis. DESIGN Prospective clinical study. SETTING University hospital, Germany. SUBJECTS 25 patients with oedematous (n = 9) or necrotising (n = 16) pancreatitis, and 20 healthy controls. MAIN OUTCOME MEASURES Concentrations of endotoxin and immunoglobulins (classes G, M, and A) directed at two lipid A molecules, four lipopolysaccharides, and alpha-haemolysin of Staphylococcus aureus measurements in plasma during a 12 day period. RESULTS There were no differences in the degree of endotoxaemia between patients with oedematous and necrotising pancreatitis on admission. However, from the day after admission and throughout the observation period patients with necrotising pancreatitis had significantly higher concentrations of endotoxin than those with oedematous pancreatitis. Concentrations of IgM specific for endotoxin peaked at day 4, and then decreased in patients with oedematous pancreatitis while remaining high for those with necrotising pancreatitis. There was only a slight increase in IgA specific for endotoxin, and IgG and immunoglobulins to gamma-haemolysin remained steady throughout the observation period. There was strong cross-reactivity (r > 0.7) between IgM specific for endotoxin (70%), but this was less with IgA (52%), and IgG (20%). CONCLUSIONS Necrotising pancreatitis is accompanied by persistent endotoxaemia with an extended rise in antiendotoxin antibodies. Patients with oedematous pancreatitis have a transient endotoxaemia with a temporary increase of Ig specific for endotoxin. Endotoxin stimulates the synthesis of specific antibodies (IgM) despite general immunosuppression.

Association of endotoxemia and production of antibodies against endotoxins after multiple injuries.

BACKGROUND Endotoxemia after injury has been a controversial issue. Endotoxins stimulate the innate and adaptive immune system. OBJECTIVE To investigate endotoxemia and its effects on the production of antiendotoxin antibodies of cultured mononuclear cells of patients with multiple injuries. METHODS Blood samples of 20 patients with multiple injuries were collected up to 12 days after trauma. The endotoxin concentration was measured in the plasma, and mononuclear cells were isolated and cultured. Specific antibodies against two lipopolysaccharides, one lipid A preparation, and alpha-hemolysin of Staphylococcus aureus were measured in the cell culture supernatant by an enzyme-linked immunosorbent assay. RESULTS Endotoxemia peaked at admission of the patients, decreasing thereafter to almost normal values within 5 days. Isolated mononuclear cells synthesized antibodies against all tested antigens with a peak at or between day 5 and day 7. The increase was significant for immunoglobulin (Ig)A and IgM specific to all endotoxins tested and for IgA specific to alpha-hemolysin. However, there were no significant changes of the concentrations of total IgM, IgA, and IgG. All specific IgG remained unaffected. CONCLUSION Patients with multiple injuries initially have temporary endotoxemia. Endotoxin may be suggested as a stimulator of the synthesis of antiendotoxin antibodies, in particular of the IgA and IgM class in patients with multiple injuries.

Demonstration of an Interaction between Transferrin and Lipopolysaccharide – An in vitro Study

Transferrin is reported to be a major lipopolysaccharide binding protein of human plasma, at least in vitro. By use of the limulus-amebocyte-lysate test the influence of transferrin on endotoxicity was studied. In the absence of any other protein human iron-free transferrin was able to strongly enhance endotoxicity in a concentration-dependent manner. Similar results were obtained when transferrin was added to primarily heat-inactivated plasma. Even in this assay the endotoxin recovery increased when transferrin was exogenously added. On the other hand, transferrin inhibited endotoxicity when inactivation of the plasma samples was performed after the addition of endotoxin and transferrin. These results lead to the conclusion that transferrin in fact interacts with lipopolysaccharide in a biologically important manner. In order to achieve neutralization of endotoxin, however, other plasma constituents are needed. The hypothetical function of transferrin is possibly a disaggregation of lipopolysaccharide micelles, following the interaction between the two molecules. The present data should justify further studies in order to clarify a possible benefit of the substitution of transferrin during gram-negative sepsis.

Endotoxin-lnduced Release of Interleukin 6 and Interleukin 1β in Human Blood Is Independent of Tumor Necrosis Factor Alpha

It has been suggested that tumor necrosis factor alpha (TNF alpha) acts not only by direct toxicity, but also as a proximal mediator which is able to induce the production of other cytokines, especially interleukin 6 (IL-6) and interleukin 1 beta (IL-1 beta). In order to test the dependence of the release of these two cytokines from leukocytes upon induction by TNF alpha, we stimulated whole blood in vitro with TNF alpha and compared the cytokine levels with those induced by endotoxin. The cytokine release was also determined after stimulation by endotoxin with added TNF alpha and by endotoxin with monoclonal antibodies against TNF alpha (anti-TNF alpha) added in order to reduce TNF alpha. Unstimulated blood samples were used as controls. The plasma levels of both IL-6 and IL-1 beta were significantly higher after stimulation with endotoxin than after stimulation with TNF alpha. TNF alpha did not induce cytokine levels significantly higher than controls. The cytokine levels were the same whether or not anti-TNF alpha was included together with the endotoxin. Plasma from samples with added anti-TNF alpha had no detectable TNF alpha. Our results indicate that the leukocyte-derived production of IL-6 and IL-1 beta in whole blood is stimulated directly by endotoxin and is not mediated by TNF alpha.