Enteral Administration of High-Fat Nutrition Before and Directly After Hemorrhagic Shock Reduces Endotoxemia and Bacterial Translocation

Abstract

Objective:

To determine whether potential enhancement of endotoxin neutralization via high-fat enteral nutrition affects endotoxemia and bacterial translocation after hemorrhage.

Summary Background Data:

Endotoxin and bacterial translocation due to gut barrier failure are important initiating events in the pathogenesis of sepsis after hemorrhage. Systemic inhibition of endotoxin activity attenuates bacterial translocation and distant organ damage. Triacylglycerol-rich lipoproteins constitute a physiological means of binding and neutralizing endotoxin effectively. We hypothesized that enhancement of triacylglycerol-rich lipoproteins via high-fat enteral nutrition would reduce endotoxemia and prevent bacterial translocation.

Methods:

A rat model of nonlethal hemorrhagic shock was used. Hemorrhagic shock (HS) rats were divided into 3 groups: rats starved overnight (HS-S); rats fed with a low-fat enteral diet (HS-LF), and rats receiving a high-fat enteral diet (HS-HF).

Results:

Circulating triacylglycerol and apolipoprotein B, reflecting the amount of triacylglycerol-rich lipoproteins, were elevated in HS-HF rats compared with both HS-S rats (P ≤ 0.005 and P ≤ 0.05, respectively) and HS-LF rats (P ≤ 0.005 and P ≤ 0.05). Circulating endotoxin was lower in HS-HF rats (7.2 ± 10.2 pg/ml) compared with both HS-S rats (29.1 ± 13.4 pg/ml, P ≤ 0.005) and HS-LF rats (29.9 ± 5.2 pg/ml, P ≤ 0.005). In line, bacterial translocation was lower in HS-HF rats (incidence 4/8 rats; median 3 [range 0–144] cfu/g) compared with both HS-S rats (8/8; 212 [60–483] cfu/g; P = 0.006), and HS-LF rats (8/8; 86 [30–209] cfu/g; P = 0.002).

Conclusion:

This study is the first to show that high-fat enteral nutrition, leading to increased plasma triacylglycerol and apolipoprotein B levels, significantly decreases endotoxemia and bacterial translocation after hemorrhage.

This study reports that physiological elevation of triacylglycerol lipoproteins by administration of a high-fat enteral nutrition just before and after hemorrhage reduces endotoxemia and bacterial translocation in a rat model. A high fat concentration in perishock-administered enteral nutrition is essential in preserving gut barrier integrity.

Lipopolysaccharide (LPS) or endotoxin, a constituent of the outer membrane of Gram-negative bacteria, is an important mediator in the pathogenesis of the sepsis syndrome after major trauma, surgery, and hemorrhage.^1,2 The incidence of sepsis has increased over the years, and a further increase is expected due to aging of the population and more complex surgery.³ Although the pathogenesis of the (late) sepsis syndrome after hemorrhage is not clear, gut barrier failure is considered to play a key role.^4,5 Several animal studies clearly show that hemorrhagic shock results in gut barrier failure leading to translocation of endotoxin and bacteria.^5–9 Bacterial toxins such as endotoxin can lead to local activation of the inflammatory system and subsequent production (locally) of inflammatory cytokines leading to a further deterioration of the gut barrier and bacterial translocation.¹⁰ Moreover, an increase of systemic endotoxin levels after hemorrhage plays an important role in the development of acute lung injury.⁸ This vicious circle of endotoxemia and bacterial translocation and subsequent acute lung injury can be interrupted by interventions that neutralize circulating endotoxin.^8,9 Several physiological defense mechanisms protect against endotoxemia such as the complement system, the coagulation cascade, the inflammatory response and lipoproteins. Lipoproteins bind and incorporate both Gram-positive and Gram-negative bacterial toxins rapidly, a process that is mediated by lipopolysaccharide binding protein (LBP) and apolipoproteins.^11,12 Detoxification of endotoxin by lipoproteins prevents endotoxin from initiating an inflammatory response. Triacylglycerol-rich lipoproteins in particular are very potent inhibitors of the bioactivity of endotoxin and protect animals against endotoxin-induced lethality.^13–16 Elevation of triacylglycerol-rich lipoproteins, like chylomicrons and very low density lipoproteins (VLDL) would thus induce an increased capacity to inhibit the bioactivity of endotoxin. Physiological elevation of triacylglycerol levels occurs after a fat meal. Chylomicrons, formed in the gut and transported along mesenteric lymphatics, are present locally in the gut in the early postprandial phase. VLDL circulates systemically and is also elevated after enteral feeding.^17,18 Therefore, high-fat enteral nutrition would theoretically be very effective to inhibit the bioactivity of enteric-derived endotoxin both locally and systemically after disruption of the gut barrier as occurs following hemorrhagic shock in an early stage. Interestingly, fasting is common in surgical patients most at risk for endotoxemia of enteric origin even though a recent meta-analysis indicates that a “nil by mouth” regimen is not beneficial in gastrointestinal surgery.¹⁹ In animal studies investigating the pathogenesis of the sepsis syndrome, animals are generally fasted overnight before trauma or hemorrhage.5,6,8,20 Bark et al²¹ reported in rats that brief fasting was associated with significantly increased bacterial translocation following hemorrhagic shock compared with fed animals, indicating the importance of enteral nutrition.

The aim of this study was to induce an increase of triacylglycerol-rich lipoproteins via high-fat enteral nutrition to enhance the natural defense mechanism against endotoxin, thereby reducing endotoxemia and bacterial translocation after hemorrhage. In our experiments, we measured circulating triacylglycerol and apoB as indicators of triacylglycerol-rich lipoproteins. Circulating endotoxin and bacterial translocation to mesenteric lymph nodes, spleen, and liver were measured as endpoints.

MATERIALS AND METHODS

Animals

The present study was performed according to the guidelines of the Animal Care Committee of the University of Maastricht and this committee approved the protocol. Healthy male Sprague-Dawley rats, weighing 301–410 g (average, 342 g) purchased from Charles River (Maastricht, the Netherlands) were housed under controlled conditions of temperature and humidity. Before the beginning of the experiments, rats were fed water and chow ad libitum.

Experimental Design

Animals were divided into 5 groups (n = 8 per group). Control rats (C) were starved for 18 hours and killed to assess the effect of fasting alone. Sham-shock (SS) rats were starved and the femoral artery was cannulated, but no shock was induced. The hemorrhagic shock groups were either starved 18 hours before the procedure (HS-S) or enterally fed with a low-fat liquid enteral diet (HS-LF) or a high-fat liquid enteral diet (HS-HF) via oral gavage. The exact set up of the procedure is displayed in Figure 1. Blood and tissue samples were taken at 24 hours after onset of shock. The low-fat diet contained 6.9% (energy-percent) proteins, 75.4% carbohydrates, and 16.7% fat. The amount of fat in the low-fat diet was isocalorical to that present in standard rodent chow. The high-fat liquid enteral diet was isocaloric and isonitrogenous to the low-fat diet, but contained 6.9% proteins, 40.9% carbohydrates, and 52.2% fat. The protein source was lean milk, and the carbohydrate source was a mixture of sucrose and corn starch. The lipid source was vegetable oil with a fatty acid composition of 8.1% saturated fatty acids (SAFA); 58.9% monounsaturated fatty acids (MUFA), of which oleic acid was the main source (57.4%); 28.2% consisted of polyunsaturated fatty acids (PUFA), of which linoleic acid was the main source (23%), the amount of n-3 and n-6 fatty acids in the high-fat nutrition was less than 5% of the total fat content. The types of carbohydrates and fat used in both diets were identical.

FIGURE 1. Experimental procedure and feeding schedule. At -18 hours, rats were starved overnight (1), 45 minutes before withdrawal of blood anesthesia was given and a femoral artery catheter inserted (2); at t = 0, hemorrhagic shock was induced (3); after 50 minutes, the femoral artery catheter was removed and the wound was closed (4); after 6 hours, all shock groups were allowed standard chow ad libitum (5); at 24 hours after (sham) shock (t = 24 hours), rats were killed (6). A liquid enteral nutrition (low-fat or high-fat) was administered via gavage in the fed groups (HS-LF and HS-HF), at −18 hours (3 mL), −2 hours (0.75 mL), −45 minutes (0.75 mL), +3 hoursv(0.75 mL), and +6 hours (0.75 mL).

Hemorrhagic Shock Procedure

Rats were anesthetized with intraperitoneally injected sodium pentobarbital (40 mg/kg). The skin over the left femoral area was shaved and desinfected with povidone iodine solution. The animals were placed in the supine position and allowed to breathe spontaneously. During surgery and throughout the experiment, body temperature was maintained at 37°C with an infrared heating lamp controlled by a thermo analyzer system (Hugo Sachs Elektronik, March-Hugstetten, Germany) connected to a rectal probe. The femoral artery was dissected using aseptic technique and cannulated with polyethylene tubing (PE-10) containing heparinized saline (10 IU/ml). Arterial blood pressure was continuously measured (Uniflow external pressure transducer; Baxter, Utrecht, the Netherlands) and recorded as mean arterial pressure (MAP). Heart rate (HR) was continuously assessed from the instantaneous pressure signal. To keep the arterial catheter patent, it was constantly perfused with physiological saline (3 mL/h) via the Uniflow system; no heparin was used. After an acclimatization period of 30 minutes, rats were subjected to hemorrhage by withdrawing blood in quantities of 2.1 mL/100 g of body weight (representing approximately 30–40% of the circulating volume) at a rate of 1 mL/min. At 50 minutes after the induction of shock, the catheter was removed and the femoral artery was ligated. Six hours after hemorrhage, the rats were allowed access to standard chow ad libitum. Rats in the SS group were anesthetized, and the left femoral artery was cannulated. SS rats were monitored similarly to the hemorrhagic shock group; however, no blood was withdrawn.

Twenty-four hours after induction of shock, the rats were anesthetized with sodium pentobarbital (60 mg/kg). The skin over the abdomen was shaved and disinfected with povidone iodine. The abdomen was opened via a midline incision, blood samples were taken, and mesenteric lymph nodes, the midsection of the spleen, and segment IV of the liver were aseptically removed for bacteriological examination.

Plasma Samples

Arterial blood samples were collected in heparinized pyrogen-free glass tubes at the time of induction of shock (t = 0) and 24 hours later. Plasma was separated by centrifugation, frozen immediately, and stored (−20°C) until the time of the assay. Hematocrit values were directly measured at the time of shock (t = 0) and 24 hours later.

Triacylglycerol and apoB

Circulating triacylglycerol was determined using a standard enzymatic assay (Sigma Diagnostics, St. Louis, MO). Levels of apoB were determined using Sandwich-ELISA with a polyclonal antibody against rat apoB, kindly provided by Dr. G. Gibbons, University of Oxford, UK. Briefly, 96-well immunomaxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with rabbit antirat apoB in a concentration of 1 μg/ml. ApoB in plasma was detected with biotin-conjugated rabbit antirat apoB followed by peroxidase-conjugated streptavidin. TMB was used as substrate for peroxidase. Plates were read in a microplate reader at 450 nm. As no standard was available, apoB levels were expressed as percentage of pooled plasma of normal healthy male Sprague-Dawley rats with the same weight (300–400 g).

Endotoxin and Bacterial Translocation

Total circulating endotoxin was determined by a chromogenic Limulus Amoebocyte Lysate (LAL) assay (Endosafe, Charles River, Charleston, SC). In short, after thawing, heparinized plasma was directly diluted 2-fold in pyrogen-free water and subsequently heated for 5 minutes at 75°C, to inactivate LPS inhibitors in plasma. LAL-reagent and plasma were incubated for 45 minutes at room temperature. After blocking the reaction with H₂SO₄, an end point measurement was used. This assay has an effective range from 0.001–1 ng/ml.

Mesenteric lymph nodes (MLN), the midsection of the spleen, and a segment of the liver were collected aseptically in 2 mL of preweighed thioglycolate broth tubes (Becton Dickinson [BBL] Microbiology Europe, Maylan, France). After weighing, the tissue specimens were homogenized with sterile grinding rods (Potter S, B.Braun Melsungen, Melsungen, Germany). Subsequently, 500-μL volumes were transferred onto the following agar plates: Columbia III blood agar base supplemented with 5% vol/vol sheep blood (BBL) (duplicate plates), Chocolate PolyviteX agar (BioMérieux, Marcy L'Etoile, France), and Schaedler Kanamycin-Vancomycin agar supplemented with 5% sheep blood (BBL). Aliquots were spread over the entire surface of the agar. All agar plates were incubated for 48 hours in a 5% CO₂-enriched atmosphere or under anaerobic conditions (Shaedler agar plates). After incubation, the colonies were counted on the nonselective Columbia sheep blood agar plates. For determination of the number of colony-forming units (cfu) per gram of tissue, the number of colonies was counted on all aerobic plates and next adjusted to the weight of the grounded tissue. All different colony types were identified to the species level using conventional techniques

Statistical Analysis

Bacterial translocation data are represented as median and range; Mean arterial pressure and heart rate as median with 25^th to 75^th percentile; other data are represented as mean ± SD. A Mann-Whitney U test was used for between-group comparisons. The χ² test was used to compare incidence of translocation. A nonparametric Spearman correlation test was used for bivariate correlations. P ≤ 0.05 was considered statistically significant.

RESULTS

Hemorrhagic Shock Procedure

The severity of the hemorrhagic shock insult as reflected by changes in mean arterial pressure (MAP), heart rate (HR), and hematocrit was similar for all 3 shock groups, ie, HS-S, HS-LF, and HS-HF (Fig. 2). Directly after induction of shock (t = 0), mean MAP-values decreased from 100 (90–110) mmHg to 26 (23–31) mmHg, and the HR decreased from 395 (369–415) beats per minute (bpm) to 200 (167–232) bpm in all 3 shock groups. Hematocrit was reduced from 42 ± 2.3% at t = 0 to 29 ± 2.3% at 24 hours after shock (t = 24 hours), P < 0.001. After 50 minutes, both MAP and HR recovered to respectively 68 (59–77) mmHg and 339 (313–368) bpm. All rats recovered spontaneously after the hemorrhage, and no deaths occurred. These data are comparable with those reported by other groups using a similar model of nonlethal hemorrhagic shock.^20,22

FIGURE 2. Mean arterial pressure (MAP) of shock rats during the observation period. MAP pressure is represented as median with 25^th–75^th percentile. MAP pressure before shock (pre) was 100 (90–110) mmHg and decreased to 26 (23–31) mmHg directly after hemorrhage (t = 0). There was no significant difference in MAP between the groups during the observation period.

Triacylglycerol and apoB

As expected, circulating triacylglycerol levels at the time of shock (t = 0) were significantly higher in HS-HF rats (184.5 ± 96.2 mg/dL) compared with both HS-LF rats (18.8 ± 14.8 mg/dL, P < 0.005) and HS-S rats (23 ± 40.7 mg/dL, P < 0.005) (Fig. 3A). There was no statistical difference between the HS-S group and the HS-LF group. After 24 hours (t = 24 hours), plasma triacylglycerol was still elevated in the HS-HF rats (87.8 ± 23.6 mg/dL), whereas plasma triacylglycerol levels were below detection level in both the HS-LF rats (P < 0.005) and the HS-F rats (P < 0.005). As shown in Figure 3B, plasma concentrations of apoB in HS-HF rats at t = 0 were higher compared with both HS-LF and HS-S rats. However, statistical significance was observed only between the HS-HF and HS-LF rats (P = 0.006). After 24 hours, circulating apoB levels in plasma were still significantly elevated in the HS-HF group compared with the HS-S group (P < 0.05), but not with the HS-LF group. As expected, circulating triacylglycerol and apoB levels were significantly correlated at both t = 0 (r = 0.471, P ≤ 0.01) and t = 24 hours (r = 0.314, P ≤ 0.05)

FIGURE 3. High-fat enteral nutrition leads to enhanced triglyceride and apoB concentrations. (A) Triglyceride concentrations in plasma were significantly increased in HS-HF rats at both t = 0 (diagonally striped bars) and 24 hours later (horizontally striped bars) compared with both HS-S and HS-LF groups. Values are expressed as mean ± SD. *P < 0.005 versus HS-S group at t = 0. **P < 0.001 versus HS-S group at t = 24 hours. ^†P < 0.005 versus HS-LF group at t = 0. ^‡P < 0.001 versus HS-LF group at t = 24 hours. (B) ApoB in the HS-HF group at t = 0 was significantly higher compared with the HS-LF group, *P = 0.006. At t = 24 hours, apoB concentrations in the HS-HF group were significantly higher compared with the HS-S group, ^†P = 0.036. Values are expressed as percentage of the apoB concentration in pooled plasma of healthy rats. Concentration of apoB in Control, Sham-Shock, fasted Hemorrhagic Shock (HS) groups, and the HS group fed with a low-fat enteral diet did not significantly differ.

Circulating Endotoxin and Bacterial Translocation

The induced hypertriglyceridemia would potentially increase capacity to inhibit bacterial toxins such as endotoxin and thus preserve gut barrier function. In the systemic circulation, endotoxin levels remained near the detection levels in the control groups (C and SS), Figure 4. Circulating endotoxin after 24 hours in the HS-HF group (7.2 ± 10.2 pg/ml) was significantly lower compared with both the HS-S group (29.1 ± 13.4 pg/ml, P = 0.005) and the HS-LF group (29.9 ± 5.2 pg/ml, P = 0.002). There was no statistical difference between the HS-S group and the HS-LF group. The bacterial translocation data are represented in Table 1. As expected, cultures from tissues taken from the control group were sterile. In the SS rats, 3 animals had positive cultures, with low numbers of bacteria. After hemorrhagic shock, bacterial translocation was demonstrated in all animals. The number of bacteria found in mesenteric lymph nodes (MLN), spleen, and liver was significantly higher in the HS-S group compared with the SS rats (P = 0.001). The median of colony-forming units found in MLN of the HS-LF group was not significantly different from the amount of bacteria found in MLN of the HS-S group (212 versus 86 cfu/g, P = 0.059). In contrast, after high-fat enteral nutrition (HS-HF), sterile cultures were found in 4 of 8 rats. In addition, the amount of bacteria found in MLN, spleen, and liver in the whole group was considerably reduced and significantly lower compared with the HS-S and the HS-LF groups. Overall, the bacteria most frequently found in the cultures were Escherichia coli, Enterococcus faecalis, Staphylococcus aureus. Additionally, Proteus and Lactobacillus species were sporadically identified. Bacteria were more often found in MLN than spleen or liver, and Enterobacteriacea translocated more frequently than Lactobacillus species. Both the species of bacteria and the frequency of translocation to the MLN are comparable with other rat studies investigating bacterial translocation after hemorrhagic shock.^20,23 In line with our hypothesis, levels of circulating triacylglycerol were inversely related with the total bacterial translocation, r = −0.346, P ≤ 0.05 (Fig. 5A) and circulating endotoxin levels, r = −0.598, P ≤ 0.01 (Fig. 5B) in each hemorrhagic shock animal.

FIGURE 4. Circulating endotoxin at 24 hours after shock. Circulating endotoxin is significantly lower in the HS-HF group compared with both the HS-S group (*P = 0.005) and the HS-LF group (^†P = 0.002). Each individual measurement is presented with the 5th and 95^th percentile and mean ± SD.

TABLE 1. Bacterial Translocation at 24 Hours After Shock

FIGURE 5. Circulating triglycerides are inversely correlated with total bacterial translocation and circulating endotoxin in all hemorrhagic shock rats. (A) Circulating triglycerides are inversely correlated with total bacterial translocation. Total bacterial translocation expressed as colony forming units (cfu) per gram (g) is plotted against circulating triglycerides (mg/dL), r = −0.346, *P ≤ 0.05. (B) Circulating triglycerides are inversely correlated with the circulating endotoxin level at t = 24 hours. Circulating endotoxin (pg/ml) is plotted against circulating triglycerides (mg/dL), r = -0.598, **P −0.01.

DISCUSSION

In the present study, we show that administration of high-fat enteral nutrition before and directly after hemorrhage induces an increase in circulating triacylglycerol and apoB concentrations. Interestingly, this study is the first to show that administration of high-fat enteral nutrition was accompanied by reduced plasma endotoxin levels and bacterial translocation. Ulevitch et al²⁴ and Tobias et al²⁵ proposed in the early 1980s a possible role for high-density lipoproteins (HDL) to bind and inactivate endotoxin. Later, others discovered that hypertriglyceridemia as a result of de novo synthesis in the liver is part of the early response to low-dose endotoxin.²⁶ This endotoxin-mediated increase in circulating triacylglycerol-rich lipoproteins is considered to have a protective function. In vitro studies showed that triacylglycerol-rich lipoproteins such as VLDL and chylomicrons are potent inhibitors of endotoxin activity.^13,27 In addition, in vivo studies showed that preincubation of endotoxin with triacylglycerol-rich lipoproteins or repeated intravenous infusions with chylomicrons protect animals against endotoxin-induced death.^13,14 The proposed mechanism for these protective properties of triacylglycerol-rich lipoproteins may be 2-fold. Firstly, VLDL and chylomicrons can directly inhibit the bioactivity of endotoxin by uptake of endotoxin into these lipoproteins. Secondly, clearance of circulating endotoxin in plasma is enhanced by increased hepatocellular uptake of endotoxin associated with chylomicrons.¹⁶ Triacylglycerol-rich lipoproteins shunt endotoxin away from Kupffer cells towards hepatocytes, thereby decreasing cytokine release and TNF-mediated inflammation. Neutralization of the bioactivity of endotoxin by lipoproteins is mediated in part by LBP, apoA-1, and apoB.^12,28,29 LBP functions as a lipid transfer molecule, catalyzing the detoxification of endotoxin. ApoB, the main apolipoprotein of triacylglycerol-rich lipoproteins, is considered to function as a binding site for both endotoxin and LBP.

In our study, an increase of triacylglycerols was observed only in the HS-HF rats at both t = 0 and t = 24 hours. Chylomicrons are primarily triacylglycerol particles, and chylomicron formation is a characteristic property of the enterocytes during the postprandial state.³⁰ Therefore, the majority of triacylglycerol measured at t = 0 is probably found in chylomicrons. As chylomicrons are rapidly cleared from the circulation,³¹ the prolonged hypertriglyceridemia found at t = 24 hours may be due to elevated VLDL levels. At the same time, also apoB levels in plasma were enhanced in the HS-HF group. Since the apoB concentration in plasma is known to be strongly correlated with circulating triacylglycerol levels,³² we expected a higher apoB concentration in plasma of HS-HF rats compared with that of HS-S and HS-LF rats. Statistical significance was obtained with the HS-LF group at t = 0 and the HS-S group after 24 hours. The absence of a postprandial increase in both triacylglycerols and apoB in the HS-LF group confirms earlier results that the postlipemic response depends on the amount of fat in the administered diet.³³ The hypertriglyceridemia and the elevated apoB levels in the HS-HF rats, which reflect an elevation of triacylglycerol-rich lipoproteins, were accompanied with lower circulating endotoxin levels and reduced bacterial translocation. This effect was specific for the high-fat nutrition, because both endotoxin levels and bacterial translocation to MLN, spleen, and liver were not significantly different in the HS-LF group compared with the HS-S group.

A transient endotoxemia, attributed to direct endotoxin translocation, has been described in a different model of hemorrhagic shock, with a peak concentration at 150 minutes after onset of shock.¹⁰ The endotoxemia that we observed at 24 hours after shock may be either the result of direct leakage due to a reduced gut barrier function or derived from translocated bacteria. At this stage, our data do not allow to distinguish between these potential sources of endotoxin. A common hypothesis proposes that endotoxin triggers an inflammatory process locally in the gut, resulting in tissue damage, leading to a deterioration of the gut barrier function.9,10,34,35 Neutralization of the bioactivity of endotoxin in an early stage would thus prevent this loss of barrier function. The importance of endotoxin neutralization after hemorrhagic shock in preserving gut barrier integrity is illustrated by animal studies showing that systemically administered endotoxin inhibitors, ie, recombinant bactericidal/permeability increasing protein (rBPI) and the endotoxin neutralizing monoclonal antibody, WN1 2225, decrease bacterial invasion into the intestinal wall.^8,9 We propose that in our study triacylglycerol-rich lipoproteins play a role in neutralizing endotoxin and that this explains the protective effect of high-fat enteral nutrition regarding bacterial translocation. This is supported by the significant negative correlation between circulating triacylglycerol and both total bacterial translocation and circulating endotoxin levels in all hemorrhagic shock rats. At present, it is unclear whether triacylglycerol-rich lipoproteins function locally in the gut or in the systemic circulation. The fact that chylomicrons, apoB, and LBP, all essential in endotoxin neutralization, are produced by enterocytes^12,36–38 is in favor of local endotoxin neutralization by high-fat enteral nutrition.

Opposed to data found in rodents, there is still controversy about the effect of triacylglycerol-rich lipoproteins on endotoxin responsiveness in humans. Van der Poll et al³⁹ showed that the in vivo response to endotoxin in humans is not inhibited by hypertriglyceridemia. However, Harris et al⁴⁰ showed that preincubation of endotoxin with triacylglycerol-rich lipoproteins attenuated the inflammatory response evolving from this toxic compound. Moreover, a recent human study from our group showed that postprandial chylomicrons are very potent in neutralizing both endotoxin and lipoteichoic acid in a lipopolysaccharide-binding protein (LBP)–dependent fashion, resulting in reduced cytokine and chemokine secretion.⁴¹ The fact that van der Poll et al used a bolus injection of endotoxin, creating a sudden increase in endotoxin levels might explain these results, because activation of leukocytes by endotoxin may be more rapid than binding of endotoxin by triacylglycerol-rich lipoproteins.^13,42 In contrast, our model creates a situation in which endotoxin gradually translocates from the gut lumen into the systemic circulation, resembling the clinical situation. In this setting, exposure of endotoxin to triacylglycerol-rich lipoproteins leading to neutralization may precede the exposure to leukocytes.

In comparison with studies on triacylglycerol-rich lipoproteins and endotoxin neutralization in humans, data on the inhibitory effect of HDL on endotoxin responsiveness are more apparent.⁴³ As HDL can be up-regulated by triacylglycerol-rich lipoproteins via cholesteryl ester transfer protein (CETP) or phospholipid transfer protein (PLTP),⁴⁴ triacylglycerol-rich lipoproteins may also indirectly contribute to protection against endotoxin in humans.

In surgery, preoperative fasting is currently still a common routine;⁴⁵ however, a recent meta-analysis of controlled clinical trials concludes that there is no clear advantage of keeping patients on a “nil by mouth” regimen.¹⁹ Short-term fasting increases the number of coliform bacteria and promotes bacterial adherence to the intestinal mucosa in rats; in a situation of gut barrier failure, these phenomena are thought to promote bacterial translocation.⁴⁶ Bark et al²¹ already observed that rats receiving enteral nutrition before hemorrhagic shock had less bacterial translocation compared with rats which were fasted for 24 hours. Also clinical studies showed the benefit of early postoperative nutrition compared with either fasting⁴⁷ or TPN^48–51. In addition, certain nutrients such as glutamine, arginine, omega-3 fatty acids, and nucleic acids have immune-enhancing effects and reduce wound complication, infection, and hospital stay when added to the enteral diet.^47,52 Our data show that a strong additional protective effect can be obtained by peri-shock administration of an enteral nutrition containing a high concentration of fat.

Taken together, our current data are the first to show that a simple and relatively short nutritional intervention just before and directly after hemorrhage with a high-fat diet results in improved gut barrier function as reflected by reduced endotoxemia and bacterial translocation after 24 hours. The observed effects seem to be largely dependent on the amount of fat in the enterally administered diet. Whether this can be attributed to early and local scavenging of endotoxin by chylomicrons, leading to less local inflammation or to the prolonged hypertriglyceridemia, which also results in systemical endotoxin neutralization, remains to be investigated. This study indicates that further studies on the potential benefit of high-fat enteral nutrition are needed.

ACKNOWLEDGMENTS

We thank M.G.A. Oude Egbrink from the Physiology Department (University of Maastricht) for facilitating our hemorrhagic shock experiments. We also thank Dr. A. van den Bogaard for his advice.