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Cardiovascular Research 2003 59(1):105-112; doi:10.1016/S0008-6363(03)00347-X
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Copyright © 2003, European Society of Cardiology

Tissue metabolism during endotoxin shock after pretreatment with monophosphoryl lipid A

Stephan Klausa,*, Karl H Staubachb, Matthias Heringlakea, Jan Gliemrothc, Peter Schmuckera and Ludger Bahlmanna

aDepartment of Anesthesiology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany
bDepartment of Surgery, Medical University of Lübeck, Lübeck, Germany
cDepartment of Neurosurgery, Medical University of Lübeck, Lübeck, Germany

stephan.klaus{at}epost.de

* Corresponding author. Tel.: +49-451-500-2766; fax: +49-451-500-3510.

Received 3 December 2002; accepted 12 March 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Preconditioning pigs with low doses of monophosphoryl lipid A (MPL), a non toxic derivate of lipid A, has been shown to induce endotoxin hyporesponsiveness and to reduce the metabolic and hemodynamic consequences of endotoxin shock. However, the mechanism is presently unclear. This study was designed to elucidate the effects of pretreatment with MPL on tissue metabolism in different organs by in vivo microdialysis of interstitial fluid. Methods: In a controlled animal study at the university research laboratory, seven female mixed-breed pigs were exposed to an endotoxin infusion (1 µg/kg b.w. per h) after pretreatment with MPL in incremental doses of endotoxin during days 5–2 before the experiments. Seven animals receiving a saline pretreatment served as a control group. Hemodynamic variables and blood gas analyses including blood lactate were determined every 30 min until the animals died. Interstitial lactate and glycerol levels were measured in muscle, subcutaneous tissue and liver using in vivo microdialysis. Results: Survival time was significantly prolonged after MPL preconditioning (8.95 (7.5–9.1) h vs. 5.35 (5.0–5.6) h, P<0.05). Hemodynamic parameters were not significantly different between the treatment and control groups, while mixed venous saturation (81% (70–93%) vs. 30% (22–48%)) and arterial blood pH (7.39 (7.33–7.44) vs. 7.21 (7.1–7.25)) and pO2 were significantly higher in the preconditioned group (P<0.05). The interstitial concentrations of lactate and glycerol in all investigated tissues were significantly higher in control animals than the those who had been pretreated with MPL (P<0.05). Conclusions: Preconditioning with low doses of monosphosphoryl lipid A attenuates the negative effects of endotoxemia on tissue metabolism, probably by reducing O2-consumption. These changes may be subtle and, hence, only fully detectable by monitoring tissue metabolism.

KEYWORDS Shock; Monitoring; Metabolism; Pigs; Sepsis; Vaccination


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Despite growing understanding of the pathophysiological mechanisms precipitating sepsis to septic shock and—subsequently—to multiorgan failure and numerous trials aimed at modulating the septic inflammatory cascade, the mortality of sepsis and septic shock remains tremendously high [1].

Endotoxic shock induced by a continuous infusion of lipopolysaccharides of Gram-negative bacteria has repeatedly been used as an animal model of septic shock and may profoundly affect tissue homeostasis. Interestingly, preconditioning with endotoxin has been shown to ameliorate the hemodynamic and metabolic sequelae of endotoxin shock; a phenomenon of endotoxin hyporesponsiveness that—in its most successful form—has been named endotoxin tolerance. Recent attention has been given to the use of non-toxic lipopolysaccharides (LPS) for induction of an endotoxin tolerance. Monophosphoryl lipid A (MPL) in animal and human studies [2] has recently been shown to modulate immunological response while having low toxic effects and being well tolerated.

No data are available about the effects of MPL preconditioning on tissue metabolism during endotoxemia. Biochemical tissue monitoring is now possible using the technique of microdialysis [3]. Microdialysis has recently been introduced in neurosurgery and diabetology as a monitoring tool that allows variations in tissue metabolism to be monitored, thereby offering the possibility to detect local metabolic derangements before systemic effects may occur [4].

Hence the present study was designed to determine the effects of MPL preconditioning on hemodynamics and on systemic and regional markers of metabolism during endotoxemia in porcine endotoxic shock.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Experimental protocol
The investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). After approval by the local institutional review board 14 female German mixed-breed pigs (body weight 26 kg (21–33 kg)) were involved in this study. Pigs were randomly assigned to a control group (n = 7) and a treatment (=preconditioned) group (n = 7). Following random selection the pigs were pretreated according to a protocol based on previous observations (unpublished data). The preconditioned group was treated with intravenous injections of MPL (Salmonella minnesota R595) on days 5, 4, 3 and 2 before the experiments, in increasing doses, 5 ng/kg on day 5, 10 ng/kg on day 4, 30 ng/kg on day 3 and finally 50 ng/kg on day 2. The control group received saline injections at the same time intervals as the treatment group. The application of MPL or saline injections was performed by an institutional veterinarian not involved in this study. On the day prior to the experiments neither group received any treatment.

Fluid intake was unrestricted while the animals were fasted overnight before the experiments. After premedication with an intramuscular injection of ketamine (20 mg/kg) and midazolam (5 mg/kg) the animals were anesthetized with etomidate (1 mg/kg). Following tracheostomy the pigs were ventilated with a fixed minute volume of 12.5 ml/kg at a rate of 12 per min (inspiratory:expiratory time ratio 1:2) and an inspiratory oxygen concentration of 30% (EVA, Fa. Dräger, Luebeck, Germany). Analgesia and sedation was maintained by continuous infusion of fentanyl (0.025 mg/kg per h) and midazolam (1.8 mg/kg per h), a bolus was applicated when appropriate as tested by the corneal reflex. Muscle relaxation was achieved with pancuronium bromide (0.1 mg/kg per h). All animals received Ringer's solution at a rate of 3 ml/kg per h adapted from previous studies [5].

An arterial catheter (18G, Fa. Vyggon, Ecouen, France) was inserted into the right femoral artery. A pulmonary artery catheter (Vigilance CCO, Baxter, USA) was inserted via the right internal jugular vein to monitor continuous cardiac output (CCO), mixed venous oxygen saturation (SvO2) and the core body temperature.

Arterial blood pressure, central venous pressure, pulmonary artery pressure and heart rate were recorded online (Sirecust 1281, Fa. Siemens/Germany).

CCO and mixed venous oxygen saturation were measured continuously via the pulmonary artery catheter. Values were recorded every 30 min after start of continuous endotoxin infusion.

SvO2 continuously was measured with the CCO catheter. Arterial blood samples were drawn every hour for analysis of paO2, paCO2, pH, arterial base excess and lactate concentrations (ABL700, Radiometer Copenhagen, Denmark).

2.2 Endotoxin infusion
Endotoxin shock was induced by administration of endotoxin from Salmonella friedenau (H 909) at a constant dose of 1 µg/kg per h until the death of the animal. Measurements were performed every 30 min. Observation period regarding the statistical calculation was focussed on the interval where all 14 animals were still alive (0–300 min, n = 7 per group). All animals surviving 9.0 h of the observation period were terminated by an overdose of midazolam, fentanyl and potassium.

2.3 Microdialysis
A paramedian laparotomy was performed and a microdialysis catheter (CMA 60, CMA/Microdialysis, Solna, Sweden) was inserted into the the right hepatic lobe and secured at the liver surface by sutures. Additional microdialysis catheters were placed into the right adductor muscle and subcutaneously in the right gluteal area. Each microdialysis catheter was connected to a perfusion pump (CMA 107; CMA-Microdialysis, Solna, Sweden) with the flow rate set to 0.5 µl/min. The catheters were perfused with lactate-free Ringer's solution and the dialysate collected in microvials for subsequent analysis.

Due to low perfusion rate and the long membrane part of the microdialysis catheters the recovery rate (ratio of concentrations dialysate/interstitium) previously was described with approximately 90% [6]. Therefore the obtained samples closely represent the actual interstitial concentrations [3]. In vivo recovery was not assessed for the investigated organs. In this study the relative changes of the metabolites to baseline values were compared to eliminate differences in tissue-dependent recovery. The metabolites of the carbohydrate and lipid metabolism (lactate and glycerol) were determined using a photometric assay (CMA 600, Solna, Sweden).

2.4 Statistical analysis
If not otherwise stated, data are presented as median+range (min–max). The basal efflux of the microdialysis analytes was calculated as the average out three consecutive samples taken before start of the endotoxin infusion and was set 100% for each curve. The subsequent results are expressed as percentage of the basal efflux. Interindividual data were analyzed with a Friedman test followed by Mann–Whitney U-test. Intraindividual data were analyzed with Wilcoxon's matched pairs test. A P value <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
No significant differences between both groups were observed at baseline values before application of the endotoxin infusion.

3.1 Survival time
Survival in the preconditioned group was significantly prolonged compared to the control group from a median of 5.35 (range 5.0–5.6) h to 8.95 (7.5–9.1) h (Fig. 1, P<0.05).


Figure 1
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  		Fig. 1 Survival-time after start of continuous endotoxin infusion demonstrating a significantly prolonged survival after pretreatment with MPL compared to the control animals. Values of *P<0.05 indicate significant difference between both groups, the data are plotted for the individual values.

 
3.2 Hemodynamics, oxygenation and blood chemistry
The results for heart rate (HR), mean arterial pressure (MAP) and cardiac output (CO) are shown in Fig. 2a–c. The differences between the two groups did not reach the level of significance. Values for mean pulmonary arterial pressure (MPAP), body temperature (BT), arterial pCO2 and pO2 are presented in Table 1 (start, 2.5 and 5 h of observation period, respectively). Apart from a higher pO2 MPL pretreatment compared to control did not induce any significant differences onto the above mentioned parameters.


Figure 2
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  		Fig. 2 (a–c) Hemodynamic parameters (heart rate, mean arterial pressure and cardiac output) after start of endotoxin infusion. No significant differences were observed between pretreated (open symbols) and control group (closed symbols) during observation period. Values of #P<0.05 indicate significant differences from baseline values at 2.5 and 5 h; values of *P<0.05 indicate significant differences between group contrasts. Data are presented as median±quartiles.

 

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  		Table 1 Values for mean pulmonary artery pressure (MPAP), body temperature (BT), arterial pO2 and arterial pCO2 during endotoxin infusion

 
A significant decrease of SvO2 down to 30% (22–48%) was observed in the control group, while the pretreated animals retained a level of 81% (70–93%) during endotoxin administration (P<0.05; Fig. 3a). Furthermore endotoxin infusion resulted in a pronounced decrease of arterial pH values in the control group (P<0.05; Fig. 3b). Parallel to this decrease, plasma lactate levels rose up to 4.2 mmol/l (1.2–6.1 mmol/l), while it remained stable after preconditioning with MPL (P<0.05, Fig. 3c).


Figure 3
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  		Fig. 3 (a–c) SvO2, arterial pH and blood lactate values of pretreated (open symbols) and control animals (closed symbols) during endotoxemia. Values of *P<0.05 indicate significant differences between group contrasts. Values of #P<0.05 indicate significant differences from baseline values at 2.5 and 5 h. Data is presented as median±quartiles.

 
3.3 Microdialysis
3.3.1 Lactate
After the start of endotoxin infusion, interstitial lactate concentrations in all tissues of the control group animals were significantly higher than in animals in the pretreated group (P<0.05). A profound and significant increase was detected in the subcutaneous tissue (392% (311–938%) of baseline), the muscle (269% (233–383%)) and the liver (332% (279–439%)) of the control group. In contrast, pretreatment with MPL in all investigated tissues ameliorated interstitial lactate accumulation and resulted in nearly unchanged levels (P<0.05; Fig. 4a–c).


Figure 4
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  		Fig. 4 (a–c) Interstitial lactate concentrations of subcutaneous tissue, muscle and liver of pretreated (open symbols) and control animals (closed symbols) during endotoxemia. Values of *P<0.05 indicate significant differences between group contrasts. Values of #P<0.05 indicate significant differences from baseline values at 2.5 and 5 h. Data are presented as median±quartiles.

 
3.3.2 Glycerol
Endotoxemia induced an increase of the glycerol values in the subcutaneous tissue of the control group to 825% (422–879%) of baseline values, while MPL pretreatment resulted in a decrease with significant lower levels (25% (16–52%); P<0.05). The relative increase in the control animals of interstitial muscular glycerol concentration was 677% (516–697%) and 449% (219–698%) in the hepatic tissue of the baseline values. Pretreated animals did not demonstrate any relevant changes of the glycerol concentration in response to endotoxin infusion and remained at significantly lower levels compared to the control group (P<0.05; Fig. 5a–c).


Figure 5
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  		Fig. 5 (a–c) Interstitial glycerol tissue concentrations in subcutaneous tissue, muscle and liver of pretreated (open symbols) and control animals (closed symbols) during endotoxemia. Values of *P<0.05 indicate significant differences between group contrasts. Values of #P<0.05 indicate significant differences from baseline values at 2.5 and 5 h. Data are presented as median±quartiles.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Preconditioning with monophosphoryl lipid A (MPL) has been shown to ameliorate the inflammatory [2] and hemodynamic effects of experimental endotoxin shock [5,7,8]. While an antagonistic effect towards endotoxin has been suggested, the mechanisms involved in this host defense are far from being clearly defined.

The present study was designed to elucidate the effects of MPL preconditioning on tissue metabolism in various organs to determine how MPL acts. Our data clearly show that pretreatment with this non-toxic lipid, ameliorates the negative effects of endotoxin shock and significantly prolongs survival time. Remarkably no significant effects of MPL on systemic hemodynamics were observed, while SvO2, blood pH and pO2 were significantly decreased in the control group and elevated in the treatment group. This suggests, that the beneficial effects of MPL to the tissue may be attributable to a decrease in cellular oxygen extraction and consumption. Alternatively these observations may be assessed as an attenuation of the increased metabolic response to endotoxemia by pretreatment with MPL. As the experiment progresses, the decrease in cardiac index—although not statistically significant—also contributes to the increase in lactate concentration and decrease in SvO2. Interestingly, using in vivo microdialysis, the effects on tissue metabolism—less increase in lactate and glycerol—were detected as changes in liver, muscle and subcutaneous tissue.

Porcine endotoxic shock is a well-established experimental model of human septic shock, typically being followed by a stimulation and upregulation of proinflammatory cytokines (TNF, IL1, IL6, IL8). After pretreatment with endotoxin or MPL, respectively, a remarkable resistance to the toxic effects of endotoxin is induced and includes a resistance to various aggressions such as lethal irradiation and hypoxia [1,9–11]. Initially after endotoxin administration, cardiac output usually increases as a consequence of a decrease in systemic vascular resistance. This is followed subsequently by a marked decrease of systemic blood flow due to a severe myocardial depression [1,12]. Microcirculatory alterations concerning a diminished and heterogeneous pattern of organ blood flow distribution, have been reported to be involved in and be responsible for the tissue malnutrition with subsequent organ dysfunction during critical illness [13].

The inflammatory and circulatory effects of experimental endotoxemia are usually accompanied by a remarkable increase in blood lactate levels and a decrease in blood pH [1]. Interestingly, increased concentrations of lactate during human septic shock have been shown to be an ominous prognostic sign. However, the mechanisms leading to this biochemical alteration are highly controversial. Possible explanations have recently been reviewed by the roundtable conference on tissue oxygenation [13]. It was stated that during critical illness limited tissue oxygenation is based on disorders in the central (cardiac output), regional (distribution of blood flow between organs) and the microregional (nutritional blood flow within the organs) circulation. This latter factor requires particular attention. Few data are available on studies using microdialysis and tissue monitoring during experimental endotoxin shock and inflammation [14–17]. Oldner et al. [15,16] observed an increase in interstitial hypoxanthine, measured in the intestinal tract—as an estimate of ATP consumption—but did not find any significant intramuscular changes of hypoxanthine levels in response to endotoxin administration. In contrast to these findings we observed a significant increase of lactate levels after induction of the endotoxin shock in liver, muscle and subcutaneous tissue. Additionally, interstitial glycerol was significantly elevated in these tissues. This end product of phospholipid degradation indicates sympathetic activation with increased lipolysis and subsequent direct damage (toxicity, oxygen radicals, etc.) of cell membranes [3,18].

Endotoxin hyporesponsiveness is a well-known phenomenon that has been described by pretreatment with endotoxin in a porcine shock model by our group [5,8]. The mechanisms active in the modulation of this host defense have been reported to involve the production of cytokines as well as reactive nitrogen intermediates and the release of superoxide and hydrogen peroxide [19]. The findings in the present study are in contrast with previous observations from Staubach et al. showing a significant higher mean arterial pressure during endotoxin shock in preconditioned animals [5]. Carpati et al. in their study were able to show a significant attenuation in the decrease in cardiac output, stroke volume index, blood pressure and mixed venous saturation associated with lethal endotoxemia by pretreatment with MPL [7]. These results contradict our observations, while it has to be mentioned that lipid A partial structural analogs give us an increased therapeutic window compared to LPS, but its toxicity and immunostimulatory activity are difficult to calculate. However, in line with our findings, higher blood lactate levels in the control animals were observed. The beneficial effects of MPL preconditioning in the present study were most obvious in the metabolic parameters determined by microdialysis. A possible reason for improvement in the metabolic parameters in the absence of hemodynamic changes has extensively been reviewed by Fink, introducing the concept of a mitochondrial dysfunction called ‘cytopathic hypoxia’ in septic conditions [20,21]. Hence, the proposed antagonistic effect of MPL towards endotoxin on the cellular level with an improvement in the metabolic parameters does not necessarily have to be associated with concomitant hemodynamic changes. This suggests, with respect to the fact that hemodynamics in both groups were not significantly different, that metabolic variations during experimental septic shock may be subclinical; and at least in an early state, only be detectable by metabolic monitoring using microdialysis.

It remains to be determined, if this also applies to the setting of human septic shock and if particular constellations in tissue metabolites might be associated with a prognosis regarding the clinical outcome. Such an association has been demonstrated by case reports of Stjernström et al. [22] and de Boer et al. [23] in a series of septic patients. Additionally the promising results of our study emphasize the role of microdialysis at a time during critical illness, when metabolic deterioration may be subclinical and, hence, in the clinical context, only detectable by this method.

Time for primary review 42 days.


   Acknowledgements
 
The authors thank Mr. Lee Reilly for proofreading the manuscript.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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