Membrane-rich milk fat diet provides protection against gastrointestinal leakiness in mice treated with lipopolysaccharide

J. Dairy Sci. 94:2201–2212 doi:10.3168/jds.2010-3886 © American Dairy Science Association®, 2011.

Membrane-rich milk fat diet provides protection against gastrointestinal leakiness in mice treated with lipopolysaccharide D. R. Snow,* R. E. Ward,*1 A. Olsen,† R. Jimenez-Flores,‡ and K. J. Hintze*1,2 *Department of Nutrition, Dietetics and Food Sciences, and †Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan 84322 ‡Dairy Products Technology Center, Department of Agriculture, California Polytechnic State University, San Luis Obispo 93407

ABSTRACT

Milk fat globule membrane is a protein-lipid complex that may strengthen the gut barrier. The main objective of this study was to assess the ability of a membrane-rich milk fat diet to promote the integrity of the gut barrier and to decrease systemic inflammation in lipopolysaccharide (LPS)-challenged mice. Animals were randomly assigned to one of 2 American Institute of Nutrition (AIN)-76A formulations differing only in fat source: control diet (corn oil) and milk fat diet (anhydrous milk fat with 10% milk fat globule membrane). Each diet contained 12% calories from fat. Mice were fed diets for 5 wk, then injected with vehicle or LPS (10 mg/kg of BW) and gavaged with dextran-fluorescein to assess gut barrier integrity. Serum was assayed for fluorescence 24 h after gavage, and 16 serum cytokines were measured to assess the inflammatory response. Gut permeability was 1.8-fold higher in LPS-challenged mice fed the control diet compared with the milk fat diet. Furthermore, mice fed the milk fat diet and injected with LPS had lower serum levels of IL-6, IL-10, IL-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-3 compared with LPS-injected mice fed the control diet. The results indicate that the membrane-rich milk fat diet decreases the inflammatory response to a systemic LPS challenge compared with corn oil, and the effect coincides with decreased gut permeability. Key words: inflammation, lipopolysaccharide (LPS), milk fat globule membrane (MFGM), phospholipid INTRODUCTION

Systemic inflammation causes increased intestinal permeability and gut barrier integrity is compromised leading to dysfunction (Zayat et al., 2008). Although the mechanisms of gut barrier dysfunction are unclear,

Received October 1, 2010. Accepted February 4, 2011. 1 These authors contributed equally to this work. 2 Corresponding author: [email protected]

it is known that pro-inflammatory cytokines, such as IL-6, IL-4, IL-13, IFN-γ, and tumor necrosis factor (TNF)-α increase the permeability of intestinal epithelia (Fink, 2003; Yang et al., 2003; Lewis and McKay, 2009; Yajima et al., 2009). Several studies suggest that milk polar lipids, including sphingomyelin and gangliosides, positively affect the gut by exhibiting a protective effect against colon cancer (Dillehay et al., 1994; Schmelz et al., 1996, 2000; Nilsson and Duan, 2006; Duan and Nilsson, 2009; Snow et al., 2010). Other studies have linked sphingolipids and other milk fat fractions containing phospholipids and gangliosides to the inhibition of a pro-inflammatory response both systemically and in the gut (Park et al., 2005b, 2007; El Alwani et al., 2006; Dalbeth et al., 2010). One interesting dietary source of polar lipids is the milk fat globule membrane (MFGM), a protein-lipid complex originating from the apical surface of mammary epithelial cells, which surrounds the fat globules in milk. During milk synthesis, triglyceride droplets are secreted from the endoplasmic reticulum, complete with a coat of polar lipids, and are thought to transit directly to the cell’s apical surface. As they are secreted from the epithelial cells into the alveolar lumen, they pass through the apical membrane and are encapsulated in the plasma membrane, complete with the exterior glycocalyx, resulting in discrete globules with 3 layers of membrane lipids (Keenan and Patton, 1995). These milk fat globules range in diameter from 0.1 to about 15 μm (Michalski et al., 2001, 2005; German and Dillard, 2006) and have a nonpolar lipid core that is surrounded by the MFGM, containing phospholipids and membrane glycoproteins (major components of MFGM are found in Table 1). While MFGM is present in all dairy products to some extent, churn buttermilk, a coproduct of butter production, is especially enriched in this membrane fraction. When milk fat is churned, the membrane surrounding the fat globules is disrupted, and the free fat is released as globules coalesce. The result of this process is a solid fat phase, butter, and an aqueous phase, churn buttermilk, which has a long history of anecdotal health-

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Table 1. Major components of the milk fat globule membrane (MFGM)1 Lipid

Amount (%)

Polar lipid

Amount (%)

Triacylglycerols Diacylglycerols Monoacylglycerols Sterols Sterol esters Free fatty acids Hydrocarbons Phospholipids

62 9 Trace 0.2–2 0.1–0.3 0.6–6 1.2 26–31

Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Lysophosphatidylcholine    

22 36 27 11 4 2    

Protein Mucin 1 Xanthine oxidoreductase PAS III CD 36 Butyrophilin PAS 6/7 (MFG-E8)2 Adipophilin Fatty acid-binding protein

1

Data from Keenan and Patton (1995). Although not listed in the original source, MFG-E8 is another name used for PAS 6/7.

2

promoting associations. Recently, it has been suggested that specific components of churn buttermilk, namely constituents of MFGM, may have interesting nutraceutical properties (Spitsberg, 2005). As more comprehensive analytical tools, such as proteomics and lipidomics, are used to characterize the MFGM, it is becoming clear that the sheer complexity of this material is impressive (Fauquant et al., 2005; Reinhardt and Lippolis, 2006; Fong et al., 2007; Cavaletto et al., 2008; Vanderghem et al., 2008). Furthermore, several groups have conducted studies to facilitate its recovery from buttermilk and other dairy streams (Astaire et al., 2003; Corredig et al., 2003; Spence et al., 2005, 2009a,b; Morin et al., 2006a,b, 2007; Rombaut et al., 2006, 2007; Rombaut and Dewettinck, 2007). Although the complexity of MFGM is well documented and methods are available to recover it from dairy processing operations, few studies have been conducted to investigate possible nutritional benefits. One interesting, and perhaps underappreciated, aspect of native milk fat globules is the size distribution and resulting surface area. In both humans (Rüegg and Blanc, 1981; Hamosh et al., 1985; Michalski et al., 2005) and bovines (Michalski et al., 2001, 2006), 2 distinct globule distributions are apparent, one with an average diameter of less than 1 μm and one centered around 4 μm. As the surface area of milk fat globules in human milk has been estimated to be over 500 cm2/mL (Rüegg and Blanc, 1981), we hypothesized that this material likely has specific interactions, both physically and biochemically, with the gut epithelia during the digestion process. In support of this hypothesis, PAS 6/7 [also known as milk fat globule–EGF factor-8/lactadherin (MFG-E8)], a protein component of MFGM, is an important endogenous factor in maintaining epithelial homeostasis and mucosal integrity of the intestinal tract. In fact, treatment of septic mice i.p. with recombinant MFG-E8 improved mucosal healing (Bu et al., 2007). Additionally, polar lipids, such as those found in MFGM, have been linked to improved resistance of epithelia to stress (Dial and Journal of Dairy Science Vol. 94 No. 5, 2011

Lichtenberger, 1984, 1987; Kivinen et al., 1995; Park et al., 2007; Dial et al., 2008). In this study, the hypothesis that a milk fat-based diet rich in MFGM would promote gut mucosal integrity was tested in Bagg albino (BALB)/c mice challenged with LPS. Systemically, LPS induces an inflammatory response mimicking gastrointestinal injury, and has been used in numerous studies to emulate the inflammatory response seen in gram-negative bacterial infections (Dial et al., 2008). Because of the complex composition of potentially bioactive proteins and polar lipids of MFGM, we hypothesized that diets providing it as a component of the diet might protect against the inflammatory response and corresponding compromised gut barrier integrity induced by LPS treatment. Furthermore, instead of using isolated components of MFGM, we specifically chose to use a relatively complex milk fat isolate from a dairy processing operation, as this material is currently being produced in large quantities around the world and is available as a potential bioactive food ingredient. MATERIALS AND METHODS MFGM Isolation

Sweet cream was obtained from Cal Poly Dairy Farm (San Luis Obispo, CA) milk using a cream separator (model 345, Alfa-Laval, Richmond, VA) after pasteurization. After a tempering period of 16 h at 4°C, the cream was churned using a continuous pilot scale butter churn (Egli AG, Bütschwil, Switzerland), and butter fines were removed by filtration through cheese cloth. A pilot plant-scale system (R-12 model, GEA-Niro Filtration, Hudson, WI) using 2 spiral polymeric membranes fitted in parallel on the module (10 kDa molecular weight cutoff, 11.33 m2 total surface area) was used for buttermilk concentration. The process was carried out at 25°C, the transmembrane pressure was 600 kPa and the feed pump was operated at 35 Hz. The ultrafiltration was conducted until a 10-fold volumetric

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concentration factor was reached. Diafiltration (DF) was done adding tap water continuously at 25°C to the feed tank to replace the removed permeate until reaching a 5-fold diafiltration factor (5 × DF). The final retentate was spray-dried (Niro Filterlab Spray-drier; GEA-Niro Filtration) to obtain buttermilk powders. Diet Formulation

Diets were formulated to differ only in the composition of the fat. Prior to the diet formulation, the protein, total fat, ash, and lactose were analyzed in the MFGM isolate, as described previously (Morin et al., 2007), and the results are shown in Table 2. To ensure that diets were similar in micronutrients, the MFGM was analyzed for specific minerals by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Based on the composition of the MFGM powder listed in Table 2, it was determined that addition of 125 g of the isolate to 1 kg of diet would provide approximately 10% of the fat as phospholipids (6 g), as well as 68 g of casein, 17 g of whey proteins, approximately 20 g of triglycerides, 5g of lactose, and 5.6 g of ash. Thus, the control diet contained 5% fat provided as corn oil, whereas the membrane-rich milk fat diet also contained 5% fat, but half of the fat was provided as anhydrous milk fat and half was provided as MFGM isolate (Tables 2 and 3). To balance the 2 diet compositions, the casein:whey ratio in the corn oil diet was adjusted by substituting 17 g of whey proteins for casein, and the carbohydrates were adjusted by substituting 5 g of lactose. The MFGM isolate contained significant levels of calcium, phosphorus, zinc, and copper, a mineral mix devoid of these components was used for the membrane-rich milk fat diet (Table 3), and specific amounts were added as listed in Table 3 to balance the compositions. Because of this process, the sucrose content of the 2 diets appears to be more different than it is (495 g for the control diet versus 477 g for the membrane-rich milk fat diet). This is due to the fact that the mineral mix of the control diet contains 11.5% sucrose and, when added at 35 g/kg of diet, it contributes 4.1 g of sucrose. Therefore, the total sucrose content of the control diet was 499g/kg. The mineral mix of the membrane-rich milk fat diet contained 73% sucrose and the zinc premix contained 99.5% sucrose, and both of these components contributed an extra 17 g of sucrose, resulting in 494 g/diet (Table 3). Although we took great care to match the nutrient profiles of the 2 diets, a slight difference in nutrient density existed, with the control diet containing 3.8 kcal/g versus 3.7 for the membrane-rich milk fat diet (Table 3). Diets were formulated by Dyets, Inc. (Bethlehem, PA). The fatty acid composition of each diet was determined us-

Table 2. Proximate composition of milk fat globule membrane (MFGM) isolate

Item

MFGM isolate (%) 2

Protein Casein Whey Fat3 Triglycerides Phospholipids Ash Lactose Water

68     20     4.5 4.0 3.5

Amount of each component provided by MFGM powder1 (g) 85 ~68 ~17 25 ~19 ~6 5.6 5.4 4.3

1 These are the estimated contributions of these components to the diet based on the addition of 125 g of buttermilk powder to the diet formulation. 2 The ratio of casein:whey is estimated based on the composition of whole milk. These values do not account for proteins of the MFGM. 3 The triglyceride:phospholipid ratio of the MFGM powder was determined to be 3:1, based on the analysis of these components in the final diet.

ing GC with flame ionization detection, as described previously (Snow et al., 2010). Study Design

The hypothesis that a membrane-rich milk fat diet confers protective effects against LPS-induced inflammation and improves gut barrier protection was tested using a 2 × 2 factorial design at 24- and 48-h time points. Weanling, male BALB/c mice were acquired from Charles River Labs (Wilmington, MA). Upon arrival to our animal facility, the mice were fed chow diets for 2 wk to acclimatize. Subsequently, mice were randomly assigned to one of the following treatments for each time point: 1) control diet (AIN-76A), saline vehicle control injection (n = 6); 2) control diet, LPS injection (n = 6); 3) membrane-rich milk fat diet, saline vehicle control injection (n = 6); 4) membrane-rich milk fat diet, LPS injection (n = 6; Figure 1). Mice were fed experimental diets for 5 wk. This time frame has previously been used in an investigation examining dietary modulation of LPS-induced inflammation (Peterson et al., 2008). On d 35, mice were injected i.p. with a dose of saline vehicle control or 10 mg of LPS/kg of BW (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO). This dose of LPS has previously been shown to induce monocyte adhesion and infiltration into intestinal tissues of mice and induce intestinal distress (Peterson et al., 2008). To assess intestinal permeability, 4,000-Da Dextran-FITC (Sigma) suspended in PBS was gavaged immediately after vehicle control or LPS injection. After injection and gavage, animals were serially killed at 24 and 48 h by CO2 asphyxiation. These time points were chosen based on data from preliminary work, wherein little Journal of Dairy Science Vol. 94 No. 5, 2011

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Table 3. Composition of diets Ingredient MFGM3 powder (g) Carbohydrate4 (g/kg of diet) Sucrose Lactose Corn starch Powdered cellulose Protein (g/kg of diet) Casein Whey dl-Methionine Fat Corn oil Anhydrous milk fat Vitamins and minerals (g/kg of diet) Vitamin mix5 Mineral mix Zinc premix (5 mg/g) Magnesium oxide Dicalcium phosphate, anhydrous Monopotassium phosphate Potassium phosphate Choline bitartrate Caloric density6 (kcal/kg)

AIN-76A1 —

Membrane-rich milk fat diet2 125

495 5 150 50

477 — 150 50

183 17 3

115 — 3

50 —

— 25

10 35 — — — — — 2 3.8

10 17.5 4.4 0.7 12.4 2.3 5.82 2 3.7

1

American Institute of Nutrition diet. To formulate the membrane-rich milk fat diet, 125 g of buttermilk powder was added to the formulation. Based on the values in Table 2, this contributed approximately 68 g of casein, approximately 17 g of whey proteins, 5 g of lactose, 25 g of fat (19 g of triglycerides, 6 g of phospholipids), and 5.6 g of ash. 3 MFGM = milk fat globule membrane. 4 Less sucrose appears to be used in the formulation of the membrane-rich milk fat diet, but the difference is closer than it appears in the table due to the technique used to balance the mineral composition. The mineral mix used for the AIN-76A diet contains 11.5% sucrose (or 4.1 g/kg of diet), whereas the mineral mix used for the membrane-rich milk fat diet contains 73% sucrose (or 12.7 g/kg of diet) and the zinc premix contains 99.5% sucrose (or 4.4 g/kg of diet). The final diets contain 499 and 494 g of total sucrose for the AIN-76A and the membrane-rich milk fat diet, respectively. 5 Due to the mineral profile of the buttermilk extract used to formulate the diet, a custom mineral mix was used to formulate the membrane-rich milk fat diet. To balance the minerals, several components were used, as listed in the table. 6 Because of the many manipulations necessary to match the diets, a slight difference in caloric density existed. 2

plasma fluorescence was measured at 0 and 12 h after LPS injection, and the peak value occurred at 24 h (data not shown). In addition, a similar study found statistically significant differences in rodent membrane integrity using a similar design (Krimsky et al., 2000). All animal experimental protocols were approved by the Utah State University Institutional Animal Care and Use Committee (IACUC). Gut Permeability and Systemic Inflammation Measurements

To assess gut barrier integrity, serum samples from all time points were assayed for dextran-FITC (Amersham Typhoon Trio+ and ImageQuant TL software; GE Healthcare Bio-Sciences, Uppsala, Sweden). Dextran-FITC permeation of the gut has shown to be a sensitive indicator of gut barrier integrity in vivo and Journal of Dairy Science Vol. 94 No. 5, 2011

appearance of serum FITC is positively correlated with intestinal insult. Serum cytokine analysis was performed on samples from all time points using the Q-Plex Mouse CytokineScreen (16-plex) array (Quansys Biosciences, Logan, UT). Cytokines analyzed include: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MCP1, IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation, normal T cell expressed and secreted (RANTES). Lipopolysaccharide has been shown to elevate serum concentrations of various cytokines in mice, specifically IL-6, IL-10, IL-12, MCP-1, IFN-γ, and TNF-α. Differences in diet consumption, BW, serum fluorescence and serum cytokine concentrations were determined by Student’s t-test. Serum cytokine data was normalized by log10 transformation.

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RESULTS

Weight and food intake was measured weekly. Dietary treatment did not significantly affect consumption or weight gain of animals (Figure 2). Measurements of serum fluorescence as a function of time and treatment are shown in Figure 3. The results indicate that the membrane-rich milk fat-based diet is significantly protective against LPS-induced gut permeability. The serum fluorescence of mice fed the milk fat diet and injected with LPS was not different at 24 h (5.92 ± 0.59 μg/mL) or 48 h (5.68 ± 0.55 μg/mL) than animals on the same diet and injected with saline (24 h; 5.80 ± 0.31 μg/mL and 48 h; 5.75 ± 0.46 μg/ mL). However, animals fed the control diet and injected with LPS had a 1.8-fold higher concentration of plasma fluorescence at 24 h (10.8 ± 0.18 μg/mL; P < 0.05) than the saline-injected animals (6.37 ± 1.29 μg/mL) on the same diet. Surprisingly, at 48 h, all animals fed the control diet and injected with LPS were dead. At 24 h, 7 of the 16 serum cytokines assayed were present in significantly lower concentrations in animals fed the milk fat diet versus the control diet (Figure 4). The difference in IL-6 was the most significant (2.24 pg of IL-6/mg of serum protein ± 0.34 vs. 4.00 pg of IL-6/mg of serum protein ± 0.42; P < 0.002). Other serum cytokines that were significantly affected were IL-10 (3.12 pg of IL-10/mg of serum protein ± 0.04 vs. 3.61 pg of IL-10/mg of serum protein ± 0.30; P < 0.04); IL-17 (2.79 pg of IL-17/mg of serum protein ± 0.60 vs. 3.71 pg of IL-17/mg of serum protein ± 0.64; P < 0.03); MCP-1 (1.90 ± pg of MCP-1/mg of serum protein 0.67 vs. 3.07 pg of MCP-1/mg of serum protein ± 0.83; P < 0.05); IFN-γ (2.87 pg of IFN-γ/ mg of serum protein ± 0.29 vs. 4.13 pg of IFN-γ/mg of serum protein ± 0.61; P < 0.025); TNF-α (2.47 fg of TNF-α/mg of serum protein ± 0.12 vs. 3.21 fg of TNF-α/mg of serum protein ± 0.40; P < 0.03); and IL12p70 (3.52 pg of IL-12p70/mg of serum protein ± 0.2 vs. 2.67 pg of IL-12p70/mg of serum protein ± 0.02). In addition, LPS-treated animals fed the control diet also had higher levels of IL-3 compared with the animals fed the milk fat diet, yet the latter values were below the detection limit and, thus, statistical determination of significance could not be measured. Because no mice fed the control diets survived at 48 h, a comparison to those fed the milk fat diet was not possible. However, it is interesting to note that of the 6 significantly affected cytokines at 24 h in the milk fat-fed animals, 3 had significantly lower concentrations at 48 h (IL-6, IL-17, and MCP-1), whereas 3 did not (IL-10, IFN-γ, and TNF-α; Figure 4). The concentrations of 9 serum cytokines were not significantly affected by the diets at 24 h: IL-1α, IL-1β, IL-2, IL-4, IL-5, MIP-1α, GM-

Figure 1. A 2 × 2 experimental design of treatments was used for each time point (24 and 48 h). Mice (n = 24) were randomly assigned to one of the following treatments: 1) control diet [American Institute of Nutrition (AIN)-76A], saline vehicle control injection (n = 6); 2) control diet, LPS injection (n = 6); 3) AIN-76A + milk fat diet, saline vehicle control injection (n = 6); or 4) AIN-76A + milk fat diet, LPS injection (n = 6).

CSF, and RANTES. In Figure 5, the data for some of the cytokines not significantly affected is shown, except for Il-2 and Il-4, which were below the detection limit. Although it was not possible to compare the treatments at 48 h in the LPS-treated animals, it is interesting to note that concentrations of IL-1β, IL-5, and RANTES were significantly lower at 48 h than at 24 h in the milk fat-fed animals. DISCUSSION

Because no significant differences were seen in feeding behavior and weight gain of mice between the 2 diets, the differences noted in gut permeability and cytokine induction do not appear to be linked to the amount of food consumed or to weight gain. The serum fluorescence of animals fed the control diet and challenged with LPS was significantly higher than that of animals fed the milk fat diet and challenged with LPS, suggesting the membrane-rich milk fat diet exerted a protective effect against gastrointestinal leakiness (Figure 3). This is supported by the observation that the serum fluorescence levels were not different from animals receiving vehicle control injections across both diets. One unexpected result in this study was the death of all animals fed the control diet and treated with LPS at the 48-h time point. This finding coincides with increased gut leakiness and expression of the inflammatory cytokines IL-6, IL-10, IL-17, MCP-1, IFN-γ, TNF-α, and IL-3 in LPS-injected animals fed the control diets compared with membrane-rich milk fat diet-fed cohorts. However, it is unclear as to whether the observed decrease in gut permeability and circulatJournal of Dairy Science Vol. 94 No. 5, 2011

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Figure 2. Effect of experimental diets on consumption (A) and total weight gain (B). Values are means (n = 36 control diet, n = 33 milk fat diet) ± SD. Experimental diets did not significantly affect consumption or weight gain.

ing inflammatory cytokine levels protected the milk fat-fed animals from death at the 48-h time point. Myriad reports exist of i.p. injection of LPS in mice to induce gastrointestinal hyperpermeability, yet methodologies differ between studies and, thus, it is difficult to compare results directly. Many factors would be anticipated to affect the results of such work including the mouse strain, LPS dosage, as well as the background diet. In addition, not all reported studies have followed animals to recovery, as often investigators are interested in characterizing molecular events in tissues available only in killed mice. For strains, no studies appear to exist that directly compare the sensitivities of different mice to LPS. However, in chemically induced colon cancer models, the selection of mouse strain can result in large differences in tumor number (Papanikolaou et Journal of Dairy Science Vol. 94 No. 5, 2011

al., 1998; Nambiar et al., 2003), suggesting that mouse strain plays a role in gut physiology. Doses as low as 0.5 and 2.0 mg/kg of i.p. administered LPS have been used to induce gut permeability in 129Sv/J mice (McCafferty et al., 2002) and C57B1/6J mice (Han et al., 2004), and thus, our use of 10 mg/kg may have been unnecessarily high. The selection of the LPS dose (10 mg/kg) and the mouse strain (BALB/c) was based on the study of Peterson et al. (2008), and according to the authors, animals were also fed the AIN-76A diet (personal communication). However, that study was terminated at 24 h, and thus, it is not clear if those animals would have succumbed to the stress as our control animals did. In a separate study conducted on BALB/c mice, Krakauer et al. (2010) determined that an approximately 4 mg/ kg dose of LPS caused 48% lethality, yet did not provide any information on the background diet. In preliminary work in our laboratory using the same mouse strain and LPS dose, no animal deaths were observed at the 48-h time point. However, animals were fed standard chow diets. A major difference in these 3 diets is the fat source, as the AIN-76A is made with corn oil, our membrane-rich milk fat diet with milk fat, and the chow diet with soybean oil. The fatty acid composition of all 3 diets is shown in Table 4 and from the data, it is clear that the essential fatty acid (EFA) composition is very different. The two 18-carbon EFA, linoleic acid (C18:2 n-6) and linolenic acid (C18:3 n-3), can be further elongated and desaturated to create highly unsaturated fatty acids such as arachidonic acid (ARA; C20:4 n-6), eicosapentaenoic acid (EPA; C20:5 n-3), and docosahexaenoic acid (DHA; C22:6 n-3; Nakamura et al., 2004). The long-chain n-6 polyunsaturated fatty acid, ARA, is a precursor for inflammatory eicosanoids, whereas the long-chain n-3 fatty acids, EPA and DHA inhibit the metabolism of ARA to proinflammatory eicosanoids and serve as weakly inflammatory or antiinflammatory eicosanoids themselves (Calder, 2008). Therefore, the dietary ratios and concentrations of n-6 and n-3 fatty acids can directly affect tissue levels of eicosanoid precursors, and subsequently, inflammatory processes. In support of this possibility, in a separate study we conducted with these 2 diets, the ARA level in red blood cells of Fisher-344 rats was significantly higher, whereas the DHA level was lower (Snow et al., 2010). As the diets used in this study had several notable differences, further studies will be necessary to determine which component, or group of components, in the membrane-rich milk fat diet were responsible for protecting the mice against LPS-induced gut permeability. The major difference in the diets was the fat composition, with the milk fat diet containing fewer polyunsaturated

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Figure 3. Effect of experimental diets and treatments on gut permeability at 24 and 48 h. The LPS-challenged mice fed milk fat diets had the same fluorescence levels as mice receiving saline vehicle control injection and the levels were significantly lower than those of LPS-challenged mice fed the control diet (*P < 0.05). Data are mean dextran-FITC concentrations (μg/mL of serum) ± SD.

fatty acids, a lower n6:n3 ratio, more polar lipids, and short-chain fatty acids. Although the diets did contain similar levels of total protein, the membrane-rich milk fat diet contained proteins originating from the MFGM. Of the main protein components of MFGM listed in Table 1, only MFG-E8 has been associated with direct effects in promoting epithelial barrier function, and that was with recombinant protein injected i.p. (Bu et al., 2007). It is important to keep in mind that the dietary MFGM proteins fed in this study were subjected to pasteurization in the production of the material and to the digestive enzymes of the mice. Thus, it is unclear to what extent the proteins may have survived intact in the gut in the active form, and how this may have provided protection. One class of MFGM proteins, the mucins, are resistant to host digestive enzymes, and may have indirectly offered protection in our study via binding and neutralizing gram-negative bacteria. Previous studies have shown that mucins are resistant to host digestion (Ward et al., 2005) and effectively bind gram-negative species such as Escherichia coli (Schroten et al., 1992). It is conceivable that the mucins may decrease the numbers of certain bacteria in the gut and thus, secondary exposure to gut-derived LPS in animals suffering from gastrointestinal hypermeability. Several previous studies, both in vitro and in vivo, have demonstrated the ability of milk-derived constituents to strengthen the gut barrier. For example, with

mammary and kidney cells in culture, Stelwagen and Ormrod (1998) showed that a component of whey from cows immunized with a multivalent bacterial vaccine prevented barrier leakiness induced by EGTA and enhanced restoration of the barrier after 24 h. According to the authors, the component responsible was primarily carbohydrate in nature. In rats subjected to heat stress, Prosser et al. (2004) demonstrated that supplementation with 0.4 g/d with either bovine colostrum or goat milk powder decreased permeability of the gut to 27 and 10% of the control values, respectively. Although the factor responsible for stimulating gut barrier resistance to heat was not identified in the colostrum or milk powers, it was not associated with either the casein or the fat fraction, as both were removed in the preparation of powders used in a preliminary cell culture experiment. In a more recent study, bovine colostrum was shown to decrease intestinal barrier damage, bacterial translocation, and the accompanying systemic inflammatory response induced by ischemia/reperfusion (I/R) injury in Sprague-Dawley rats when compared with saline and skim milk (Choi et al., 2009). Unlike the previous studies, the colostrum used as a treatment in their work was applied after the gut injury was induced via the I/R stress. Taken together, these data, along with the results of our study, indicate that certain components of milk may have the ability to protect the gut barrier against stress-induced leakiness and also may have the Journal of Dairy Science Vol. 94 No. 5, 2011

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Figure 4. Significant effects of dietary treatment on serum cytokine levels after LPS challenge at 24 and 48 h. All animals fed the control diet and challenged with LPS died before the 48-h time point. IL-6 (A), IL-10 (B), IL-17 (C), monocyte chemotactic protein (MCP)-1 (D), IFNγ (E), tumor necrosis factor (TNF)-α (F), and IL-12p70 (G) all had significantly decreased serum concentrations 24 h after LPS challenge. IL-3 (H) serum concentrations were below the detection limit after the 24- and 48-h time points. Data are log10 transformed mean cytokine concentrations per milligram of serum protein ± SD. The significance of the differences from the control diet (24 h) was *P < 0.05 and **P < 0.005. Journal of Dairy Science Vol. 94 No. 5, 2011

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Figure 5. Effects of dietary treatment on serum cytokine levels after LPS challenge at 24 and 48 h. All animals fed the control diet and challenged with LPS died before the 48-h time point. IL-1α (A), IL-1β (B), IL-5 (C), macrophage inflammatory protein (MIP)-1α (D), granulocytemacrophage colony-stimulating factor (GM-CSF; E), and regulated on activation, normal T cell expressed and secreted (RANTES; F) did not have significant changes in serum concentrations 24 h after LPS challenge. Data are log10 transformed mean cytokine concentrations per milligram of serum protein ± SD. The significance of the differences from the control diet (24 h) was **P < 0.005.

ability to decrease the systemic damage that occurs from a dysfunctional gut barrier. Interestingly, in newborn infants, the gut barrier is not fully developed and is permeable to intact sugars and proteins (Weaver et

al., 1984), and thus, one can hypothesize that components of milk might specifically improve gut function. Of all the components that were present in the membrane-rich milk fat diet, but not the control diet, Journal of Dairy Science Vol. 94 No. 5, 2011

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Table 4. Fatty acid composition (%) of diets Fatty acid1 C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C15:0 C16:0 C16:1n7 C18:0 C18:1n7 trans C18:1n9 C18:2n6 C18:2n3 Calories from PUFA4 (%) n6:n3 ratio

AIN-76A2

Membrane-rich milk fat diet

Chow diet

n.d.3 n.d. n.d. n.d. n.d. 0.5 0.1 11.4 0.2 2.2 0.1 27.4 56.4 1.0 6.9 56.4

2.7 2.5 1.6 3.3 3.3 10.3 1.0 27.4 1.6 11.5 2.7 23.6 4.8 0.5 0.6 9.4

n.d. n.d. n.d. n.d. n.d. 1.0 0.1 15.1 1.7 3.3 n.d. 19.3 42.9 4.9 9.5 6.4

1

Only fatty acid contributions >1.0% to at least one diet are shown. American Institute of Nutrition diet. 3 Not detected. 4 The % calories from polyunsaturated fatty acids (PUFA) was determined using the equation: %cal from PUFA = % PUFA × % of calories from fat. 2

and may have provided the protection, the polar lipids have been the most studied. The barrier properties of the gut are provided, in part, by the surface mucous that lines the gastrointestinal tract and is rich in hydrophobic lipid constituents such as phospholipids. Intraperitoneal LPS injection has been demonstrated to compromise gut barrier integrity by decreasing the surface hydrophobicity of the gut epithelia (Dial et al., 2002). This decrease in gut surface hydrophobicity is thought to be mediated through bile salts (Dial et al., 2002) and increased activity of secretory phospholipase A2 (sPLA2) in the gastric and ileal lumens (Zayat et al., 2008). Elevated expression of sPLA2 in the gut increases membrane phospholipid hydrolysis, resulting in the release of lysophospolipids and free fatty acids and ultimately results in compromised gut barrier integrity. Interestingly, it appears that exogenous polar lipids may decrease these effects. According to Dial et al. (2008), orally administered phosphatidylcholine (PC; 100 mg/kg) in rats, when provided 1 h before i.p. injection of LPS, significantly decreases the permeability of both gastric and ileal tissues, yet does not prevent the systemic TNF-α response or the increase in gastric fluid volume. The membrane-rich milk fat diet used in the study described here contained approximately 0.6% total polar lipids compared with 0.09% found in the control diet; however, unlike the study of Dial et al. (2008), in which the exogenous PC was fed acutely, the mice in our study received the membrane-rich diet for the length of the study.

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2WKHU&RPSRQHQWVRI0)*0

Another polar lipid fraction of the MFGM that affects barrier properties of the gut is the gangliosides. Park et al. (2005a) found that supplementing a rodent diet for 2 wk with 0.1% of a ganglioside-rich lipid isolate from bovine milk led to significantly more ganglioside in the intestinal mucosa and to a decrease in the cholesterol:ganglioside ratio. Using a similar experimental design in rats, this group has also shown that dietary gangliosides significantly decrease the mucosal and systemic inflammatory response to LPS injected i.p. and protects the degradation of the tight junction protein, occludin (Park et al., 2007). The results of this study indicate that the inflammatory response in the animals fed the control diet was significantly greater than in those fed the milk fat diet. The decreased serum levels of several cytokines in mice fed the experimental diet suggest some component of the milk fat diet decreased the inflammatory response following stress. However, based on our experimental design and the data collected, it is unclear how the gut leakiness and inflammation are related. Our results are consistent with those investigating the effects of dietary polar lipids on inflammation, suggesting that the high polar lipid content of MFGM could be responsible for the lowered systemic inflammatory response. For example, using both a cell culture and a mouse model of acute gout, Dalbeth et al. (2010) found that a milk fat extract, rich in phospholipids and gangliosides,

PROTECTION OF GUT BARRIER BY MILK FRACTION

exhibited dose-dependent effects in preventing cytokine secretion (cell culture) and peritonitis (mouse model). Interestingly, Treede et al. (2007) have shown in vitro in Caco-2 cells that exogenous phosphatidylcholine exerts at least part of its anti-inflammatory effect by suppressing activation of NF-κB by TNF-α, and inhibiting translocation of this transcription factor to the nucleus, which subsequently decreases downstream activation of further inflammation. In fact, when both PC and lyso-PC were provided to the Caco-2 cells along with TNF-α, the expression of several cytokines was attenuated, whereas exogenous phosphatidylethanolamine had no effect. Therefore, the inflammatory response in the mice fed the membrane-rich milk fat diet may have been attenuated due to the effects of the dietary PC. The results of this study indicate that replacing corn oil in the AIN-76A diet with a membrane-rich milk fat isolate significantly improves the barrier properties of the gut in mice subjected to LPS stress and also attenuates the systemic inflammatory response. Our results are consistent with other studies in rodents, which have documented the efficacy of polar lipids in protecting the gut barrier against stress as well as decreasing systemic inflammation, and yet we cannot rule out contribution from other components. Although the diets used in this study were formulated to differ only in the fat source, the use of a complex material like MFGM makes it impossible to associate the stress protection with any single component. Conversely, MFGM is hypothesized to contain many constituents with beneficial bioactivity (Spitsberg, 2005) and is available as an ingredient to potentially modulate the barrier properties of the gut without the necessity of isolating specific components. ACKNOWLEDGMENTS

We thank Kent Udy (Utah State University, Logan) for assistance with animal studies. Financial support for this project was provided by Dairy Management Inc. (Rosemont, IL) and by the Utah Agricultural Experiment Station (Logan). This paper was approved by the Utah Agricultural Experiment Station as paper #8161. REFERENCES Astaire, J. C., R. Ward, J. B. German, and R. Jimenez-Flores. 2003. Concentration of polar MFGM lipids from buttermilk by microfiltration and supercritical fluid extraction. J. Dairy Sci. 86:2297– 2307. Bu, H.-F., X.-L. Zuo, X. Wang, M. A. Ensslin, V. Koti, W. Hsueh, A. S. Raymond, B. D. Shur, and X.-D. Tan. 2007. Milk fat globuleEGF factor 8/lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J. Clin. Invest. 117:3673– 3683. Calder, P. C. 2008. Polyunsaturated fatty acids, inflammatory processes and inflammatory bowel diseases. Mol. Nutr. Food Res. 52:885–897.

2211

Cavaletto, M., M. G. Giuffrida, and A. Conti. 2008. Milk fat globule membrane components—A proteomic approach. Adv. Exp. Med. Biol. 606:129–141. Choi, H. S., K. H. Jung, S. C. Lee, S. V. Yim, J. H. Chung, Y. W. Kim, W. K. Jeon, H. P. Hong, Y. G. Ko, C. H. Kim, K. H. Jang, and S. A. Kang. 2009. Bovine colostrum prevents bacterial translocation in an intestinal ischemia/reperfusion-injured rat model. J. Med. Food 12:37–46. Corredig, M., R. R. Roesch, and D. G. Dalgleish. 2003. Production of a novel ingredient from buttermilk. J. Dairy Sci. 86:2744–2750. Dalbeth, N., E. Gracey, B. Pool, K. Callon, F. M. McQueen, J. Cornish, A. MacGibbon, and K. Palmano. 2010. Identification of dairy fractions with anti-inflammatory properties in models of acute gout. Ann Rheum. Dis. 69:766–769. Dial, E. J., and L. M. Lichtenberger. 1984. A role for milk phospholipids in protection against gastric acid. Studies in adult and suckling rats. Gastroenterology 87:379–385. Dial, E. J., and L. M. Lichtenberger. 1987. Milk protection against experimental ulcerogenesis in rats. Dig. Dis. Sci. 32:1145–1150. Dial, E. J., J. J. Romero, X. Villa, D. W. Mercer, and L. M. Lichtenberger. 2002. Lipopolysaccharide-induced gastrointestinal injury in rats: Role of surface hydrophobicity and bile salts. Shock 17:77–80. Dial, E. J., M. Zayat, M. Lopez-Storey, D. Tran, and L. Lichtenberger. 2008. Oral phosphatidylcholine preserves the gastrointestinal mucosal barrier during LPS-induced inflammation. Shock 30:729–733. Dillehay, D. L., S. K. Webb, E.-M. Schmelz, and A. H. Merrill Jr. 1994. Dietary sphingomyelin inhibits 1,2-dimethylhydrazine-induced colon cancer in CF1 mice. J. Nutr. 124:615–620. Duan, R.-D., and A. Nilsson. 2009. Metabolism of sphingolipids in the gut and its relation to inflammation and cancer development. Prog. Lipid Res. 48:62–72. El Alwani, M., B. X. Wu, L. M. Obeid, and Y. A. Hannun. 2006. Bioactive sphingolipids in the modulation of the inflammatory response. Pharmacol. Ther. 112:171–183. Fauquant, C., V. Briard, N. Leconte, and M.-C. Michalski. 2005. Differently sized native milk fat globules separated by microfiltration: Fatty acid composition of the milk fat globule membrane and triglyceride core. Eur. J. Lipid Sci. Technol. 107:80–86. Fink, M. P. 2003. Intestinal epithelial hyperpermeability: update on the pathogenesis of gut mucosal barrier dysfunction in critical illness. Curr. Opin. Crit. Care 9:143–151. Fong, B., C. Norris, and A. MacGibbon. 2007. Protein and lipid composition of bovine milk-fat-globule membrane. Int. Dairy J. 17:275–288. German, J. B., and C. J. Dillard. 2006. Composition, structure and absorption of milk lipids: A source of energy, fat-soluble nutrients and bioactive molecules. Crit. Rev. Food Sci. Nutr. 46:57–92. Hamosh, M., J. Bitman, D. L. Wood, P. Hamosh, and N. R. Mehta. 1985. Lipids in milk and the first steps in their digestion. Pediatrics 75:146–150. Han, X., M. P. Fink, R. Yang, and R. L. Delude. 2004. Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice. Shock 21:261–270. Keenan, T. W., and S. Patton. 1995. The structure of milk: Implications for sampling and storage A. The milk lipid globular membrane. Pages 5–44 in Handbook of Milk Composition. R. G. Jensen, ed. Academic Press, San Diego, CA. Kivinen, A., S. Tarpila, T. Kiviluoto, H. Mustonen, and E. Kivilaakso. 1995. Milk and egg phospholipids act as protective surfactants against luminal acid in Necturus gastric mucosa. Aliment. Pharmacol. Ther. 9:685–691. Krakauer, T., M. Buckley, and D. Fisher. 2010. Murine models of staphylococcal enterotoxin B-induced toxic shock. Mil. Med. 175:917–922. Krimsky, M., A. Dagan, L. Aptekar, M. Ligumsky, and S. Yedgar. 2000. Assessment of intestinal permeability in rats by permeation of inulin-fluorescein. J. Basic Clin. Physiol. Pharmacol. 11:143– 153.

Journal of Dairy Science Vol. 94 No. 5, 2011

2212

SNOW ET AL.

Lewis, K., and D. M. McKay. 2009. Metabolic stress evokes decreases in epithelial barrier function. Ann. N. Y. Acad. Sci. 1165:327–337. McCafferty, D. M., A. W. Craig, Y. A. Senis, and P. A. Greer. 2002. Absence of Fer protein-tyrosine kinase exacerbates leukocyte recruitment in response to endotoxin. J. Immunol. 168:4930–4935. Michalski, M.-C., V. Briard, and F. Michel. 2001. Optical parameters of milk fat globules for laser light scattering measurements. Lait 81:787–796. Michalski, M. C., V. Briard, F. Michel, F. Tasson, and P. Poulain. 2005. Size distribution of fat globules in human colostrum, breast milk, and infant formula. J. Dairy Sci. 88:1927–1940. Michalski, M. C., N. Leconte, V. Briard-Bion, J. Fauquant, J. L. Maubois, and H. Goudédranche. 2006. Microfiltration of raw whole milk to select fractions with different fat globule size distributions: Process optimization and analysis. J. Dairy Sci. 89:3778–3790. Morin, P., M. Britten, R. Jimenez-Flores, and Y. Pouliot. 2007. Microfiltration of buttermilk and washed cream buttermilk for concentration of milk fat globule membrane components. J. Dairy Sci. 90:2132–2140. Morin, P., R. Jimenez-Flores, and Y. Pouliot. 2006a. Effect of processing on the composition and structure of buttermilk and of its milk fat globule membranes. J. Anim. Sci. 84 (Suppl. 1):317. (Abstr.) Morin, P., Y. Pouliot, and R. Jimenez-Flores. 2006b. A comparative study of the fractionation of regular buttermilk and whey buttermilk by microfiltration. J. Food Eng. 77:521–528. Nakamura, M. T., Y. Cheon, Y. Li, and T. Y. Nara. 2004. Mechanisms of regulation of gene expression by fatty acids. Lipids 39:1077– 1083. Nambiar, P. R., G. Girnun, N. A. Lillo, K. Guda, H. E. Whiteley, and D. W. Rosenberg. 2003. Preliminary analysis of azoxymethane induced colon tumors in inbred mice commonly used as transgenic/ knockout progenitors. Int. J. Oncol. 22:145–150. Nilsson, A., and R. D. Duan. 2006. Absorption and lipoprotein transport of sphingomyelin. J. Lipid Res. 47:154–171. Papanikolaou, A., Q.-S. Wang, D. A. Delker, and D. W. Rosenberg. 1998. Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility. Cancer Lett. 130:29– 34. Park, E. J., M. Suh, K. Ramanujam, K. Steiner, D. Begg, and M. T. Clandinin. 2005a. Diet-induced changes in membrane gangliosides in rat intestinal mucosa, plasma, and brain. J. Pediatr. Gastroenterol. Nutr. 40:487–495. Park, E. J., M. Suh, B. Thomson, D. W. Ma, K. Ramanujam, A. B. Thomson, and M. T. Clandinin. 2007. Dietary ganglioside inhibits acute inflammatory signals in intestinal mucosa and blood induced by systemic inflammation of Escherichia coli lipopolysaccharide. Shock 28:112–117. Park, E. J., M. Suh, B. Thomson, A. B. Thomson, K. S. Ramanujam, and M. T. Clandinin. 2005b. Dietary ganglioside decreases cholesterol content, caveolin expression and inflammatory mediators in rat intestinal microdomains. Glycobiology 15:935–942. Peterson, D. G., A. G. Scrimgeour, J. P. McClung, and E. A. Koutsos. 2008. Moderate zinc restriction affects intestinal health and immune function in lipopolysaccharide-challenged mice. J. Nutr. Biochem. 19:193–199. Prosser, C., K. Stelwagen, R. Cummins, P. Guerin, N. Gill, and C. Milne. 2004. Reduction in heat-induced gastrointestinal hyperpermeability in rats by bovine colostrum and goat milk powders. J. Appl. Physiol. 96:650–654. Reinhardt, T. A., and J. D. Lippolis. 2006. Bovine milk fat globule membrane proteome. J. Dairy Res. 73:406–416. Rombaut, R., V. Dejonckheere, and K. Dewettinck. 2006. Microfiltration of butter serum upon casein micelle destabilization. J. Dairy Sci. 89:1915–1925. Rombaut, R., V. Dejonckheere, and K. Dewettinck. 2007. Filtration of milk fat globule membrane fragments from acid buttermilk cheese whey. J. Dairy Sci. 90:1662–1673.

Journal of Dairy Science Vol. 94 No. 5, 2011

Rombaut, R., and K. Dewettinck. 2007. Thermocalcic aggregation of milk fat globule membrane fragments from acid buttermilk cheese whey. J. Dairy Sci. 90:2665–2674. Rüegg, M., and B. Blanc. 1981. The fat globule size distribution in human milk. Biochim. Biophys. Acta 666:7–14. Schmelz, E. M., D. L. Dillehay, S. K. Webb, A. Reiter, J. Adams, and A. H. Merrill Jr. 1996. Sphingomyelin consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: Implications for dietary sphingolipids and colon carcinogenesis. Cancer Res. 56:4936–4941. Schmelz, E. M., M. C. Sullards, D. L. Dillehay, and A. H. Merrill Jr. 2000. Colonic cell proliferation and aberrant crypt foci formation are inhibited by dairy glycosphingolipids in 1, 2-dimethylhydrazine-treated CF1 mice. J. Nutr. 130:522–527. Schroten, H., F. G. Hanisch, R. Plogmann, J. Hacker, G. Uhlenbruck, R. Nobis-Bosch, and V. Wahn. 1992. Inhibition of adhesion of Sfimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: A novel aspect of the protective function of mucins in the nonimmunoglobulin fraction. Infect. Immun. 60:2893–2899. Snow, D. R., R. Jimenez-Flores, R. E. Ward, J. Cambell, M. J. Young, I. Nemere, and K. J. Hintze. 2010. Dietary milk fat globule membrane reduces the incidence of aberrant crypt foci in Fischer-344 rats. J. Agric. Food Chem. 58:2157–2163. Spence, A., J. Yee, M. Qian, and R. Jimenez-Flores. 2005. Concentration of polar MFGM lipids from buttermilk using supercritical carbon dioxide. J. Anim. Sci. 83 (Suppl. 1):144. Spence, A. J., R. Jimenez-Flores, M. Qian, and L. Goddik. 2009a. The influence of temperature and pressure factors in supercritical fluid extraction for optimizing nonpolar lipid extraction from buttermilk powder. J. Dairy Sci. 92:458–468. Spence, A. J., R. Jimenez-Flores, M. Qian, and L. Goddik. 2009b. Phospholipid enrichment in sweet and whey cream buttermilk powders using supercritical fluid extraction. J. Dairy Sci. 92:2373–2381. Spitsberg, V. L. 2005. Invited review: Bovine milk fat globule membrane as a potential nutraceutical. J. Dairy Sci. 88:2289–2294. Stelwagen, K., and D. J. Ormrod. 1998. An anti-inflammatory component derived from milk of hyperimmunised cows reduces tight junction permeability in vitro. Inflamm. Res. 47:384–388. Treede, I., A. Braun, R. Sparla, M. Kühnel, T. Giese, J. R. Turner, E. Anes, H. Kulaksiz, J. Füllekrug, W. Stremmel, G. Griffiths, and R. Ehehalt. 2007. Anti-inflammatory effects of phosphatidylcholine. J. Biol. Chem. 282:27155–27164. Vanderghem, C., C. Blecker, S. Danthine, C. Deroanne, E. Haubruge, F. Guillonneau, E. De Pauw, and F. Francis. 2008. Proteome analysis of the bovine milk fat globule: Enhancement of membrane purification. Int. Dairy J. 18:885–893. Ward, R. E., J. B. German, and M. Corredig. 2005. Composition, applications, fractionation, technological and nutritional significance of milk fat globule membrane material. Pages 213–244 in Advanced Dairy Chemistry-2. Lipids. Vol. 2. P. F. Fox and P. L. H. McSweeney, ed. Klewer Academic Publishers, New York, NY. Weaver, L. T., M. F. Laker, and R. Nelson. 1984. Intestinal permeability in the newborn. Arch. Dis. Child. 59:236–241. Yajima, S., H. Morisaki, R. Serita, T. Suzuki, N. Katori, T. Asahara, K. Nomoto, F. Kobayashi, A. Ishizaka, and J. Takeda. 2009. Tumor necrosis factor-alpha mediates hyperglycemia-augmented gut barrier dysfunction in endotoxemia. Crit. Care Med. 37:1024–1030. Yang, R., X. Han, T. Uchiyama, S. K. Watkins, A. Yaguchi, R. L. Delude, and M. P. Fink. 2003. IL-6 is essential for development of gut barrier dysfunction after hemorrhagic shock and resuscitation in mice. Am. J. Physiol. Gastrointest. Liver Physiol. 285:G621– G629. Zayat, M., L. M. Lichtenberger, and E. J. Dial. 2008. Pathophysiology of LPS-induced gastrointestinal injury in the rat: Role of secretory phospholipase A2. Shock 30:206–211.