INTRODUCTION
More than 20 years after the introduction of the "gut as motor of sepsis" hypothesis (1, 2) there is still no consensus concerning the relation between systemic inflammation and intestinal permeability. Most studies were conducted with differential sugar absorption tests to measure intestinal permeability in critically ill patients. In these tests, two sugar probes are orally administered and passively absorbed. It is assumed that absorption of the smaller molecule is relatively constant, whereas absorption of the larger molecule is influenced by alterations in intestinal permeability. However, it was recently shown that several confounders occurring in clinical practice may have contributed to the inconclusive results of permeability studies (3, 4). This seems to represent the main reason why many clinical studies have yielded conflicting results concerning the relation between severity of disease or incidence of infectious complications and intestinal permeability (5).
In animal sepsis models, both gastrointestinal mucosal perfusion deficits and systemic inflammation were found to be associated with a decrease in gut barrier function. In rodent studies, increased intestinal permeability was shown to enhance and sustain systemic inflammation by facilitating bacterial translocation (2). In addition, inflammation was found to induce or sustain increased intestinal permeability (6, 7). The relation between systemic inflammation and intestinal permeability has not been tested in humans.
Intestinal permeability can be assessed by the urinary recovery of orally administrated polyethylene glycols (PEGs). Using a mixture of PEGs with molecular masses 400, 1,500, 4,000, and 10,000 offers the possibility to assess size-dependent variations in intestinal permeability (8, 9). The molecular size of PEG 10,000 is thought to represent the size of bacterial products such as LPS (10). Thus, PEGs allow a broader range of molecular weight, thereby possibly providing more information regarding the changes in intestinal permeability. Polyethylene glycols are not therapeutically applied or endogenously produced in contrast to several components of differential sugar absorption tests, so that recovery is not influenced by administration of packed red blood cells or mannitol (3, 4).
It has been demonstrated previously that acute systemic inflammation can be induced by a low-dose infusion of Escherichia coli LPS in healthy volunteers (11), as a model of the pathophysiological changes observed in septic patients, resulting in, for example, cardiac dysfunction (12), vascular and endothelial dysfunction (13, 14), coagulation abnormalities (15), and other subclinical end-organ dysfunction (16).
The present study addresses three questions: 1) Does experimental endotoxemia resulting in systemic inflammation induce an increase in intestinal permeability in humans? 2) Are the kinetics of urinary recovery of PEGs altered during experimental endotoxemia? 3) Is increased intestinal permeability the result of inflammation or damage (ischemic injury) of enterocytes?
MATERIALS AND METHODS
Subjects
The local ethics committee of the Radboud University Nijmegen Medical Centre approved the study protocol, and written informed consent was obtained from all 14 subjects who participated in the experiments that were part of a larger endotoxin trial (NCT 00184990). Volunteers participated in a project concerning the development of LPS tolerance. During the first day, the present intestinal permeability experiments were performed. All subjects tested HIV and hepatitis B negative and did not have any febrile illness in the 2 weeks preceding the study. Subjects refrained from caffeine, alcohol, and food 10 h before the experiment. Individuals who were taking prescription drugs (except oral contraceptives) or aspirin or other nonsteroid anti-inflammatory drugs were excluded. After inclusion, baseline hemodynamic and immunological parameters were determined. Intestinal permeability was assessed by a PEG test as described in the succeeding sentences.
Experimental human endotoxemia
Subjects were intravenously injected with a bolus of 2 ng/kg body weight E. coli O:113 LPS (United States Pharmacopia, Rockville, Md). Before the LPS administration, volunteers were prehydrated with 1.5 L glucose 2.5%/NaCl 0.45%. During the first 6 h of the experiment, subjects received 150 mL/h glucose 2.5%/NaCl 0.45%, which was reduced to 75 mL/h during the next 16 h. The course of symptom score (headache, nausea, shivering, and muscle and back pain) was rated on a six-point Likert scale (0 = no symptoms to 5 = very severe symptoms), resulting in a cumulative symptom score. Blood and urine samples were taken just before LPS injection and serially thereafter up to 22 h. Routine hematology parameters were determined using flow cytometry (Sysmex XE-2100; Goffin Meyvis, Etten-Leur, the Netherlands). TNF-α, IL-6, IL-10, and IFN-γ were measured in one batch using a multiplex Luminex Assay (Luminex, Austin, Tex).
Intestinal permeability and PEG analysis
After overnight fasting, the subjects drank the PEG solution the day before endotoxin infusion and again the next day 1 h before the administration of LPS. No food intake was allowed during 6 h after ingestion. Urine was collected in portions from 0 to 2, 2 to 4, 4 to 8, and 8 to 22 h after PEG administration, and urine volumes were recorded. Polyethylene glycol recovery was analyzed by reversed-phase high-performance liquid chromatography. Urine creatinine concentrations were measured by routine clinical chemical analysis. Although it is generally accepted in current literature to provide urinary PEG recovery levels as a percentage of the ingested amount, we chose to provide a ratio between PEGs and creatinine for results concerning the kinetics of PEG recovery. Creatinine levels were measured to correct the PEG excretion for any possible changes in urinary excretion after the LPS infusion.
Polyethylene glycols with Mr 400, Mr 1,500, and Mr 4,000 were obtained from Bufa Chemical Company (Uitgeest, The Netherlands) and PEG with Mr 10,000 from Sigma Chemical Company (St. Louis, Mo). The PEG solution contained 5 g PEG 400, 1.5 g PEG 1,500, 5 g PEG 4,000, and 10 g PEG 10,000 dissolved in 100 mL water (8). Sorbate (0.1%) was added as a preservative. The PEG solutions were prepared and quality- and purity-controlled by the Department of Pharmacy of the UMC Utrecht.
Aliquots of 12 mL of each urine portion were transferred to separate plastic tubes and stored at −20°C. Upon analysis, urine samples were homogenized, and 10 mL was centrifuged at 1,000 × g for 10 min. Clear supernatant (2 mL) was desalted by treatment with an ion-exchange resin (Bio-Rad RG 501-X8, Hercules, Calif). The resin was removed by centrifugation, and 50 μL supernatant was analyzed by reversed-phase high-performance liquid chromatography (Shimadzu SCL-10A VP, Kyoto, Japan) using a 25-cm 5-μm Lichrospher 100-RP 18E column equipped with a 1.5-cm similar guard column (Li Chro Cart 2504 mm, Merck KgaA, Darmstadt, Germany) and evaporative light-scattering detection (Alltech 500). The mobile phase consisted of a gradient of 40% to 80% methanol in water. Urine creatinine concentrations were measured by routine clinical chemical analysis using a Synchron CX4 random-access multianalyzer (Beckman Instruments Inc., Brea, Calif). Detection limits were 0.05 mg/mL for PEG 400 and 0.005 mg/mL for PEGs 1,500, 4,000, and 10,000. Analytical recovery during repeated measurements of standard solutions of all four PEGs was 100% ± 4% and reproducibility was 97% ± 1%.
Determination of intestinal fatty acid binding protein
Enhanced intestinal permeability may result from enterocyte damage (death or ischemic injury). This proposed mechanism for bacterial translocation has been extensively studied. Since the last decade, intestinal fatty acid binding protein (I-FABP) has been commercially available {Derikx, 2007 822 /id}. This protein is specifically associated with enterocyte death {Derikx, 2008 823 /id}. Therefore, the possible presence of enterocyte death was evaluated by the release of I-FABP into urine as previously described (17) {Kanda, 1992 91 /id}. Human I-FABP sandwich enzyme-linked immunosorbent assay was obtained from HyCult Biotechnology BV (Uden, the Netherlands) with a detection limit of 20 pg/mL.
Statistical analysis
Power analysis was based on clinically relevant changes in the urinary excretion of PEG. In a previous study using this method, we found an SD ranging from 40% to 50%. An increase in the urinary excretion of PEG of 40% was considered clinically relevant. With an estimated SD of 50% and α = 0.05, we calculated that a sample size of 10 to 14 individuals per group would be needed to achieve a power of 80% to 90%. Therefore, 14 individuals were included. All data were analyzed using SPSS version 13.0 software (The Apache Software Production 2007, Chicago, Ill). Results are expressed by mean ± SEM. Data obtained before and during LPS infusion were compared with a paired sample t test. Repeated measurements were analyzed using two-way ANOVA. Correlation analysis was performed using Pearson product moment correlation coefficient after confirming the normal distribution of the data. Statistical significance was defined as a P value less than 0.05.
RESULTS
Experimental human endotoxemia
The Table illustrates the demographic characteristics of the healthy volunteers. All 14 subjects fulfilled the systemic inflammatory response syndrome criteria after administration of LPS. The onset of flulike symptoms began approximately 1 h after LPS administration. Symptoms were at their maximum 90 min after LPS infusion and disappeared within 8 h. MAP decreased from 103 ± 6 to 91 ± 5 mmHg (P < 0.001), and mean heart rate increased from 64 ± 4 to 95 ± 6 (P < 0.001) beats per minute at 1.5 h after infusion. Body temperature was increased after LPS from 36.0°C ± 0.1°C to a maximum of 37.8°C ± 0.1°C (P < 0.001; Table). At baseline, all cytokine levels were below the detection limit in all subjects and showed a significant increase in all volunteers during endotoxemia (Fig. 1).
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TABLE 1: Inflammatory changes of subjects during LPS infusion
Fig. 1: Cytokine response after LPS infusion. Diamonds indicate IL-6;squares, IL-10; triangles, IFN-γ; and circles, TNF-α levels during endotoxemia.
Intestinal permeability
Of the 14 participants, 2 subjects lost a urine sample the day before LPS infusion, another urine sample was lost after LPS infusion, and a third subject vomited 30 min after LPS infusion. For this reason, paired analysis could be performed on the results of 10 subjects.
The 24-h recovery of the control parameter PEG 400 did not change during experimental endotoxemia. In contrast, the 24-h recovery of PEG 1,500 and PEG 4,000 was significantly increased during endotoxemia (Fig. 2). The most important recovery of PEGs 400, 1,500, and 4,000 was observed during the first 4 h after administration of the PEG solution (Fig. 3) both in the absence and presence of endotoxemia. Polyethylene glycol 10,000 was not recovered in any of the subjects either before or after LPS infusion.
Fig. 2: Individual alterations and group means in intestinal permeability measured by the urinary excretion of PEG. Changes in PEG recovery per subject before and after LPS infusion. Of the 14 tested subjects, 10 provided samples for paired analysis for PEG recovery (mg/24 h) of PEG 400 (A), PEG 1,500 (B), PEG 4,000 (C), and PEG 10,000 (D). Blocks and bars represent mean ± SEM.
Fig. 3: Kinetics of PEG recovery before and after LPS infusion. Visualization of recovery kinetics of PEG in milligrams per millimole creatinine in the urine per time period; <2, 2 to 4, 4 to 8, and 8 to 22 h. Although the pattern of excretion did not change after LPS infusion, significant differences were found in the kinetics before and after LPS. The increased recovery of PEG 1,500 and PEG 4,000 was due to the increased recovery in the first hours after infusion of LPS (and the PEG solution was administered). After 24 h, PEG recovery returned to zero. Closed squares indicate the samples before LPS infusion; open squares, the samples after LPS infusion. PEG 400 (A), PEG 1,500 (B), PEG 4,000 (C), and PEG 10,000 (D).
Twenty-four-hour I-FABP concentrations did not change before (0.0 ± 2.8 pg/mmol creatinine) and during endotoxemia (0.9 ± 3.5 pg/mmol creatinine; P = 0.105). In addition, I-FABP concentrations per 3 h did not change after endotoxemia, thereby indicating that there was no relevant enterocyte ischemia during experimental endotoxemia.
Correlation between systemic inflammation and intestinal permeability
There was no significant correlation between the maximum IL-6, IFN-γ, or TNF-α concentrations and the observed increase in the urinary excretion of PEG 1,500 and 4,000. However, maximum concentrations of IL-10 after LPS infusion correlated significantly with the increase in intestinal permeability (Pearson correlation, r = 0.48; P = 0.027), determined by the ratio of PEG 1,500 recovery (mg/24 h) before and after LPS infusion (Fig. 4).
Fig. 4: Correlation between IL-10max concentrations and increase in urinary PEG 1,500 recovery. The increase in PEG 1,500 was significantly correlated with the maximum IL-10 levels (Pearson P = 0.027; r = 0.476).
DISCUSSION
In this study, we investigated intestinal permeability before and during systemic inflammation induced by experimental endotoxemia in healthy subjects. We demonstrated that the urinary excretion of PEGs increased 1.5- to 5-fold during endotoxemia, indicating an enhanced intestinal permeability during systemic inflammation in humans in vivo. Because the LPS-induced hemodynamic changes were only mild and the urinary excretion of I-FABP was not increased after the LPS infusion, the observed increase in intestinal permeability is most likely caused by an inflammation-induced increase in paracellular permeability and not by ischemia of enterocytes.
During endotoxemia, all subjects developed signs of systemic inflammation and showed elevated cytokine levels, of which IL-10 correlated with the increase in intestinal permeability, which adds to the notion that the degree of the innate immune response accounts for the increase in intestinal permeability.
To our knowledge, only 2 human endotoxemia studies focusing on intestinal permeability have been performed previously. After endotoxin administration, systemic absorption and excretion of lactulose increased almost 2-fold, whereas mannitol absorption and excretion increased with only 16% (18). Although intestinal permeability seemed to increase after the endotoxin challenge, no immunological parameters were analyzed in this study. In addition, lactulose was administered twice during the endotoxin challenge, which could have hampered the results during the second measuring period. Recently, large-intestine cumulative permeability of 99mTc-DTPA was assessed in six healthy subjects after endotoxin administration. No histopathological changes in the mucosal biopsies from the rectum were found, and although colorectal permeability tended to increase, this effect did not reach statistical significance in this small group (19). Nevertheless, it seems plausible that during systemic inflammation caused by experimental endotoxemia, dysfunction in barrier integrity occurs in the whole intestinal tract. Therefore, increased intestinal permeability should be suspected in all patients with severe inflammation. Consequently, a vicious circle of inflammation, increased intestinal permeability, leading to toxic mediator release, resulting in a further increase in intestinal permeability, could be generated.
Using a differential sugar absorption test, several studies have focused on intestinal permeability in patients with sepsis (3, 20-22). However, a well-conducted study of Oudemans-van Straaten et al. (3) showed that a differential sugar absorption test based on cellobiose and mannitol is not feasible in patients with organ failure. In this study, alterations in intestinal permeability could not be related to the course of disease due to treatment-related confounding factors. The described problems were also present in a heterogeneous population of trauma patients (4). Thus, a differential sugar absorption test is not an adequate detection method for intestinal permeability in critically ill patients. In contrast, the PEG test will not be affected by the administration of its compounds for therapeutic reasons, and its compounds are not produced endogenously. Analysis of intestinal permeability by PEGs demonstrated a close relation with severity of disease in patients with pancreatitis (23) but has not been tested in patients who have inflammation of an infectious cause. In addition, up to now, PEG 10,000 has not been used in a clinical setting. We are currently investigating the applicability of the PEG solution used in this present study for analysis of intestinal permeability in patients with pancreatitis, severe sepsis, and a heterogeneous population of surgical intensive care patients.
Despite the lack of adequate investigation methods for the quantification of intestinal permeability, an etiologic role for bacterial translocation in the development of organ failure has been suggested from the results of therapeutic trials. For example, several studies have demonstrated that enteral feeding reduces the incidence and severity of infectious complications in critically ill patients (24). It was suggested that this effect is at least partly due to maintaining the integrity of the gastrointestinal barrier.
Our study demonstrates indeed that intestinal permeability is increased during a state of inflammation, which may contribute to bacterial translocation. In accordance, LPS can be detected in the blood of most patients with sepsis, independent of the Gram-positive or Gram-negative nature of the infecting organism, possibly due to the absorption of LPS derived from Gram-negative flora of the gastrointestinal tract (25, 26). Translocation of bacteria or bacterial products can add to or sustain the systemic inflammatory response.
We found a correlation between maximum serum IL-10 concentrations and the increase in intestinal permeability measured by PEG recovery. This could be an indirect effect of inflammation with I/R injury alteration in mucosal blood flow or generation of other mediators independent from circulating cytokines. Although the increase in intestinal permeability correlated with IL-10, surprisingly, it did not correlate with the proinflammatory cytokines TNF-α, IFN-γ, and IL-6. This may be explained by the fact that LPS specifically induces IL-10 release by immunological cells. In vitro studies have shown that large amounts of IL-10 are produced by T lymphocytes, dendritic cells, monocytes, and macrophages in response to LPS (27). Similar to the results of our study, changes in serum IL-10 concentrations have been correlated with LPS concentrations in serum (14). In addition, IL-10 serum concentrations have been correlated with vascular permeability after LPS infusion (28), and high IL-10 levels are associated with fatal outcome in febrile patients with community-acquired infection (29), whereas these associations with proinflammatory cytokines are more difficult to obtain. Thus, although IL-10 is an anti-inflammatory cytokine, the IL-10 response seems to accurately represent the individual innate immune response and may therefore correlate with deleterious effects in end-organs that are thought to be mediated by proinflammatory cytokines or other mediators of the innate immune response. In both animal models and patient studies, severe polymicrobial sepsis evoked a strong anti-inflammatory response (30, 31). In addition, in those studies, it has been demonstrated that overexpression of IL-10 was correlated with improved intestinal permeability and outcome. It was hypothesized that a more anti-inflammatory response leads to less additional tissue damage and therefore an improved outcome. We do not state that IL-10 and intestinal permeability are causally related; however, the initial IL-10 increase during endotoxemia seems to be usable as a readout of the inflammatory response to LPS infusion. Therefore, we conclude that the severity of inflammatory response is correlated with the increase in intestinal permeability.
The findings of increased intestinal permeability after LPS infusion in this study are not caused by random physiological alterations. In a feasibility study, we showed that the intraindividual recovery of PEG 400, 1,500, and 4,000 over 24 h varied less than 10% when measured again after a 2-week interval (unpublished data). The reliability of the PEG test has been shown previously in patients with severe pancreatitis (23). Moreover, in our study, each subject served as his own control. In our view, adequate power and use of a more reliable measure of intestinal permeability strengthens the results of our study.
CONCLUSIONS
We demonstrated a correlation between the degree of systemic inflammation and an increase in intestinal permeability. Furthermore, we showed that this increase in permeability was not caused by enterocyte ischemia, but rather by inflammation-induced paracellular changes. Thus, systemic inflammation may sustain itself due to its effects on the gut. In addition, it should be noted that this human endotoxemia model is, by design, a transient state of reversible, moderate hemodynamic stress. In prolonged septic shock, enterocyte injury, apoptosis, and focal necrosis may indeed occur in addition to the increased permeability characteristics observed in this study.
ACKNOWLEDGMENTS
The authors thank Trees Jansen for cytokine measurements and research nurse Tijn Bouw for help with the conduct of the experiments.
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