Effect of Milk Fat Globule Membrane (MFGM) on Gut Barrier Protection in Runners
Condition: Inflammation
MeSH terms: Inflammation
Study type: Interventional

Summary: 
      This study was designed to determine if an ingredient in milk fat can prevent gut leakiness
      induced by running at 80% maximum oxygen consumption (VO2max). In mouse studies, the milk fat
      ingredient, and individual components thereof, have been shown to reduce gut leakiness
      induced by intra peritoneal lipopolysaccharide (LPS) injections.

      The investigators hypothesis is as follows: Compared to a sports drink with identical macro
      nutrient composition, a sports drink with milk fat globule membrane (MFGM) will reduce the
      change in gut leakiness induced by running at 90% VO2max for 30 minutes. The decrease in gut
      permeability change will be associated with a decreased area under the curve of plasma
      endotoxin for 5 hours following the exercise. In addition, decreased gut permeability will be
      associated with lower area under the curve values for inflammatory cytokines.
    

Detailed description: 
      A randomized, double-blind cross-over exercise trial was conducted with 14 runners on Utah
      State University campus. This population of 14 runners was selected from a larger population
      of 20 that was recruited to complete two initial trials. In two preliminary visits, the
      parameters necessary for conducting the experimental trial were determined. During the first
      visit, a baseline value was determined for gut permeability as well as aerobic capacity of
      runners. In the second visit the capacity to complete a run at 80% VO2max was tested and the
      effect of the exercise stress on gut permeability, plasma LPS and core temperature were
      measured.

      Once a test population was selected, a randomized, double-blind, cross-over trial was
      conducted in which MFGM was tested for the ability to prevent a change in the exercise
      induced gut permeability.

      During each testing session heart rate (HR), work output and core temperature (CT) were
      monitored. HR and CT were monitored by a CorTemp™ temperature pill which was ingested by the
      athletes prior to the trial (http://www.hqinc.net/pages/pill_page.html). In addition, blood
      was drawn at baseline (before depletion trial), immediately after the time trial, and again
      at 1h and 5h. Blood was analyzed for indices of training intensity (lactate), muscle damage
      (creatine kinase, cytokines), and gut barrier dysfunction (lipopolysaccharide, cytokines). In
      addition, immediately after the run athletes consumed a beverage with two carbohydrates
      (lactulose, and rhamnose). During the following six hours urine was collected and these
      sugars were measured via gas chromatography to determine the permeability of the gut.

      Subject Recruitment Twenty, runners (ages 18-50) were initially recruited via campus flyers,
      advertisements in the local paper and weekly sports guide. Participants who had medical
      histories including: heart disease, uncontrolled hypertension, diabetes, Crohn's Disease,
      irritable bowel syndrome, colitis, Celiac disease, inflammatory or autoimmune diseases or
      lactose intolerance were excluded from the study. All subjects were instructed to avoid all
      anti-inflammatory medications for at least 24 h prior to any testing activity. On the morning
      of the first visit, approved subjects had body composition measured by BodPod (Life
      Measurement, Inc., Concord, CA). Participants were asked to refrain from any heavy exercise
      for 48 hrs before the first visit. Subjects were given a 50 ml tube containing the 5 g
      lactulose and 2 g rhamnose sugar probe and instructed to drink the probe 4hrs after they have
      finished breakfast to determine baseline gut permeability. Subjects were given a urine
      collection container and instructed to collect all urine for 6 hrs after ingestion of the
      probe. Subjects were not to eat lunch until 1.5 hrs after drinking the sugar probe. After 6
      hrs of urine collection, subjects returned to the test site and performed a running VO2 max
      test.

      Second Visit (approximately one week after first visit) Approximately 3 hrs after completion
      of breakfast, subjects reported to the test site to have blood drawn (to determine baseline
      plasma LPS), weight measured and to ingest a temperature probe. Participants started the
      exercise challenge (60 minute run at 80% VO2 max) approximately 4 hrs after completion of
      breakfast. The exercise challenge was conducted in a climate-controlled room at 22 °C without
      a fan. Subjects were monitored to ensure they were running at 80% VO2 max and were not
      allowed to consume water during the challenge. Immediately after the exercise challenge,
      participants consumed the sugar probes. Weight was measured, blood drawn (post-exercise
      plasma LPS) and subjects were instructed to collect their urine for the next six hrs. To
      facilitate re hydration and urine production, subjects were instructed to replace 150% of
      their fluid losses (pre to post exercise weight) by water intake.

      After screening 20 subjects, a population of fifteen subjects agreed to participate in the
      cross-over trial. One subject dropped out due to a pregnancy, and four subjects could
      participate due to scheduling conflicts. One subject dropped out after sustaining a broken
      ankle that was not related to the study.

      The third and fourth visits were the crossover portion of the experiment comparing MFGM to
      the vehicle. The 3rd and 4th visits were identical to the 2nd visit except participants drank
      either the fluid replacement vehicle or fluid replacement + MFGM 1 hr before the exercise
      challenge and again immediately following the exercise. Participants and personnel
      administering experimental protocols were blinded to treatments by use of coded,
      non-transparent drink containers. Subjects complete the 4th visit one week later and consume
      the vehicle beverage or the vehicle + MFGM.

      For the crossover portion of the study, athletes were provided one of two beverages. The
      control beverage was formulated according to current sports nutrition guidelines for both
      pre- and post-workout nutrient delivery. It contained whey protein, sucrose, maltodextrins,
      chocolate flavoring and a small amount of butter oil. The test beverage was identical on a
      macro nutrient content, but contained a milk based ingredient called 'milk fat globule
      membrane.' This material is isolated from a byproduct of butter production, and is rich in
      phospholipids. The beverages were provided three hours prior to the exercise trial as well as
      immediately after. The test beverage contained a powder rich in MFGM that is produced by Arla
      Foods (Lacprodan PL-20) which contains roughly 20% phospholipids and 60% protein. In studies
      with mice, Dial et al have noted effects on gut protection in 1 h when animals are provided
      with phospholipids at 100mg/kg body weight. The second beverage was matched for macronutrient
      content, but did not contain the phospholipid powder.

      Endpoints measured Change in gut Permeability To determine resting, baseline intestinal
      permeability the method of Pals was used. This method determines intestinal permeability by
      supplying the non-digestable sugars lactulose, and rhamnose and estimates intestinal and
      gastric permeability by recovery of these sugar probes in the urine. In healthy gut
      epithelia, lactulose has very limited transport across the gut barrier. Rhamnose crosses the
      small intestinal epithelia via a transcellular route and serves as internal control to
      account for gastric emptying rate, intestinal transit time and renal function. The larger
      lactulose molecule can only cross gut epithelia via a paracellular route through tight
      junctions. Therefore a higher ratio of lactulose to rhamnose recovered in the urine indicates
      compromised intestinal barrier function. Exercise stress has been demonstrated to
      significantly increase the urinary lactulose/rhamnose ratio . Similar to our proposed
      exercise stress model, this method has been used to demonstrate that running at 80% VO2max
      for an hour doubles gut permeability compared to baseline.

      Change in plasma LPS: To assess endotoxemia, plasma LPS concentrations were measured via a
      kinetic activity as described by Nieman. Plasma LPS was measured from blood taken before
      exercise (baseline), immediately following, and at 1 and 5 hours after the exercise trial.
      Plasma LPS has been demonstrated to increase after endurance exercise bouts and is
      hypothesized to be the result of aberrant bacterial translocation of gut bacteria through
      compromised gut barrier. Elevated plasma LPS from baseline immediately after exercise has
      been demonstrated previously after a half-marathon, full marathon. It was anticipated that
      plasma LPS levels would increase after exercise stress and will be correlated to core body
      temperature and the lactulose/rhamnose index of gut barrier integrity.

      Change in inflammatory Cytokines: Plasma levels of IL-6, TNFα, IL-10, IL-17, INFγ, IL-3,
      MCP-1, IL-15 and GMCSF were measured in the baseline and in the 1 h post exercise samples.
      Cytokine concentrations were determined by multiplex enzyme-linked immunosorbent assay
      (ELISA) by a commercial vendor (Quansys Biosystems, Logan, UT). These cytokines were selected
      based on a previous study investigating gut leakiness and MFGM consumption in an
      LPS-stimulated gut leakiness trial with mice. All cytokines were significantly upregulated in
      animals with leaky guts except GMCSF, and were significantly lowered in MFGM-fed animals
      compared to controls. Moreover, Ng et al demonstrated increased plasma levels of IL-10 and
      IL-6 (50% and 65.2% increase from baseline respectively) in runners immediately following a
      half marathon. Similarly, plasma levels of IL-6 and TNFα have been demonstrated to be
      increased from baseline immediately after a marathon. Based on the preliminary data and the
      aforementioned studies, it was anticipated that increased levels of inflammatory cytokines
      would be measured after the exercise challenge and it was hypothesized MFGM would abrogate
      this effect compared to the other recovery beverages.

      Change in plasma creatine kinase: Creatine kinase was measured in samples taken before,
      immediately after and 1 hours after the exercise challenge. Creatine kinase was measured by
      using a commercial colorimetric kit (Sigma) in a 96-well UV/vis plate reader (Molecular
      Devices). Increased plasma creatine kinase is a well-established index of exercise induced
      muscle damage. In a similar study, cyclists riding at 85% VO2 max until exhaustion after
      glycogen depletion the previous day had less plasma creatine kinase when they consumed
      chocolate milk as a recovery drink compared to a carbohydrate replacement beverage.

      Plasma Lactate: To assess anaerobic metabolism, plasma lactate will be measured at baseline,
      immediately after and 2 hours subsequent the exercise stress. Lactate will be measured using
      an enzymatic test kit (r-Biopharm, Marshall, MI).

      The primary endpoint measured in this study was the lactulose/rhamnose ratios in urine and
      plasma LPS. The data were analyzed by single factor ANOVA and significant differences were
      explored with post-hoc testing. Core body temperature was measured in real time and was
      correlated with gut hypoxia and with gut leakiness.

      All components measured in the blood (lactate, LPS, cytokines, and CK) were analyzed by a two
      factor ANOVA with time and treatment as variables. Lactate was included as it is generally
      considered to be an index of anaerobic activity. One other parameter were interested in is
      creatine kinase. As mentioned in the introduction, this enzyme is detected in the blood after
      intense exercise and is thought to correlate with damage to muscles. Furthermore, at least
      two studies with chocolate milk have noted that plasma CK is lower in athletes drinking
      chocolate milk vs. other recovery drinks.

      Management plan All three PIs were actively involved in the planning and execution of this
      study, as well as in the sample processing and the analysis. Dr. Ward was responsible for
      formulation of the MFGM beverage. In addition, Dr. Ward was responsible for the GC-FID
      analysis of the urine. In addition, Dr. Ward will supervised a graduate student supported by
      this project.

      Dr. Bressel recruited the athletes and helped with organizing the tests. In addition, Dr.
      Bressel supervised all of the performance trials and the collection of the exercise data.

      Dr. Hintze was in charge of the blood and urine samples. The student (and perhaps Dr. Hintze)
      conducted the LPS the lactate and the CK assays. In addition, Dr. Hintze was responsible for
      proper sample handing of the blood and for the specimen delivery to Quansys Biosystems for
      the cytokine analysis.

      All three PIs collaborated on the data analysis and on manuscript preparation.
    

Intervention name(s): Milk fat globule membrane
Intervention type(s): Dietary Supplement
Intervention MeSH terms: Milk fat globule
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Prevention
Keywords: Gut permeability; Cytokine; Endotoxin