Milk Beta Caseins on Digestive Discomfort, Gastric Emptying

A1 and A2 β-casein are genetic variants of β-casein in cow's milk that differ by a single amino acid at position 67: histidine in A1 β-casein and proline in A2 β-casein. This structural difference can influence proteolytic cleavage during gastrointestinal digestion. Digestion of A1 β-casein may result in greater release of beta-casomorphin-7 (BCM-7), an opioid peptide that can interact with opioid receptors in the gastrointestinal tract and may influence gastrointestinal function.

Gastric emptying may contribute to gastrointestinal symptoms in lactose maldigesters by affecting the rate at which lactose is delivered to the small intestine. Previous work from our group using magnetic resonance imaging (MRI) demonstrated faster gastric emptying following consumption of A1/A2 milk compared with A1 protein-free (A1PF) milk in lactose maldigesters. More rapid gastric emptying may increase the rate of lactose delivery to the small intestine and potentially increase the amount of unabsorbed lactose reaching the colon, where microbial fermentation can contribute to gas production and gastrointestinal symptoms.

One potential mechanism underlying differences in gastrointestinal responses to β-casein variants involves opioid receptor signaling. Because BCM-7 has opioid activity, opioid receptor blockade provides an approach for investigating whether this pathway contributes to differences in gastric emptying following consumption of A1/A2 milk and A1PF milk.

This randomized, masked, crossover study evaluated gastric emptying following consumption of A1/A2 milk and A1PF milk during administration of 2 mg oral naloxone, an opioid receptor antagonist. Lactose maldigesters consumed each milk treatment during separate intervention visits. Gastric volume was assessed using MRI at baseline and at 10, 30, 60, and 120 minutes following milk consumption. Gastrointestinal symptoms were also assessed during the intervention visits.

The study was designed to determine whether differences in gastric emptying between A1/A2 milk and A1PF milk persisted during opioid receptor blockade, thereby evaluating the potential contribution of opioid receptor signaling to the gastric emptying response.

• A1/A2 milk with oral naloxone (Other): Participants consumed A1/A2 milk at a volume calculated to provide 0.5 g lactose/kg body weight, using the formula: milk volume (mL) = \[0.5 g lactose × body weight (kg) / 11 g lactose\] × 245 mL milk. Participants also received 2 mg oral naloxone during the MRI intervention visit.
• A1PF milk with oral naloxone (Other): Participants consumed A1/A2 milk at a volume calculated to provide 0.5 g lactose/kg body weight, using the formula: milk volume (mL) = \[0.5 g lactose × body weight (kg) / 11 g lactose\] × 245 mL milk. Participants also received 2 mg oral naloxone during the MRI intervention visit.

Primary outcomes

• Gastric volume measured by MRI [Baseline and 10, 30, 60, and 120 minutes after milk consumption during each of the two intervention visits]: Gastric volume was measured in milliliters (mL) using magnetic resonance imaging (MRI). Images of the stomach were acquired at baseline and at 10, 30, 60, and 120 minutes following consumption of A1/A2 milk or A1 protein-free (A1PF) milk with 2 mg oral naloxone. Gastric volume was determined from the MRI images to assess gastric emptying over the 120-minute postprandial period.

Secondary outcomes

• Gastrointestinal symptom severity [Baseline and 30, 60, 90, and 120 minutes after milk consumption during each of the two intervention visits]: Gastrointestinal symptoms were assessed using participant-reported symptom ratings at baseline and at 30, 60, 90, and 120 minutes following consumption of A1/A2 milk or A1 protein-free (A1PF) milk with 2 mg oral naloxone during each intervention visit.

Sex: All, ages: 18 Years to 65 Years

Inclusion Criteria:

* Ability to provide informed consent.
* Aged between 18 and 65 years (inclusive) at the time of screening.
* Self-identified as milk intolerant or milk avoider.
* Agree to refrain from all treatments and products for milk intolerance (e.g., laxatives, stool softeners, Pepto Bismol®, Lactaid® Dietary Supplements) during the study.
* Willing to return for all study visits and complete all study-related procedures.
* Understand and provide written informed consent in English.

Exclusion Criteria:

* Any known allergy to Naloxone.
* Allergic to milk.
* Currently pregnant, lactating or considering pregnancy.
* Cigarette smoking or other use of tobacco or nicotine containing products within 3 months of screening.
* Diagnosed with any of the following disorders associated with abnormal gastrointestinal motility, such as: gastroparesis, amyloidosis, neuromuscular diseases (e.g., Parkinson's disease), collagen vascular diseases, alcoholism, uremia, malnutrition, untreated hypothyroidism.
* History of surgery that alters the normal function of the gastrointestinal tract, including:

gastrointestinal bypass surgery, bariatric surgery, gastric banding, vagotomy, fundoplication, pyloroplasty. (Note: A history of uncomplicated abdominal surgeries, such as appendix removal more than 12 months before screening, will not be excluded).

* Past or present diagnosis of any of the following: organ transplant, chronic pancreatitis or pancreatic insufficiency, symptomatic biliary disease, celiac disease, chronic constipation, diverticulosis, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), small intestine bacterial overgrowth syndrome, gastro-esophageal reflux disease (GERD), irritable bowel syndrome (IBS), any other medical condition with symptoms that could confound data collection (e.g., gas, diarrhea, bloating, cramps, stomach pain, fecal urgency).
* Diagnosed with active ulcers, or history of severe ulcers.
* Diagnosed with diabetes mellitus (type 1 and type 2)
* Diagnosed with Congestive Heart Failure (CHF)
* Diagnosed with Human Immunodeficiency Virus (HIV), Hepatitis B or Hepatitis C
* Weigh less than 16.5 kg or have a BMI greater than 35 kg/m².
* Had recent bowel preparation for endoscopic or radiologic investigation within 4 weeks of screening (e.g., colonoscopy prep).
* Have used treatments for milk intolerance (e.g., laxatives, stool softeners, Pepto Bismol®, Lactaid®) within 7 days of screening.
* Chronic antacid or proton pump inhibitor (PPI) use.
* Used systemic antibiotics within 30 days prior to screening (you may participate one month after discontinuing antibiotics).
* Used a high colonic enema within 30 days prior to screening. 8MAY2025 IRB-2024-731 Page 3 of 11
* Had symptoms or diseases that interfere with the assessment of milk intolerance symptoms (e.g., gas, diarrhea, bloating, cramps, stomach pain, fecal urgency).
* History of alcohol abuse.
* History of opioid drug abuse (e.g., oxycodone, hydrocodone, heroin, fentanyl, morphine).
* Currently undergoing chemotherapy.
* Have used any investigational drug or participated in an investigational study within 30 days prior to screening.
* Have any cardiac pacemaker, any type of prosthesis (eye, penile), implanted cardiac defibrillator, heart valve prosthesis/stents, aneurysm clip, shunt (spinal/intraventricular), neuro or bone stimulator, wire sutures or surgical staples, insulin or infusion pump, bone/joint pin, screw, nail, plate, implanted drug infusion device, body tattoos, cochlear, otologic or ear implant, tattooed makeup (eyeliner, lip, etc.), prostate radiation seeds, breast tissue expander, IUD (intrauterine device), hearing aids, transdermal medicine patch (Nitro), body piercing(s), any metallic implants or objects, internal electrodes or wires**
* Have claustrophobia (fear of closed spaces)
* Cannot lie still for up to 75 minutes for any reason (e.g., movement disorder, severe cold with coughing).
* Worked with metal (grinding, fabrication, etc.) or had an injury to the eye involving a metallic object (e.g., metallic slivers, foreign body), injured by a metallic object that may NOT have been completely removed (e.g., bullets, shrapnel, BBs), had surgery or any similar invasive procedure, had a reaction to a contrast medium used for MRI or CT, diagnosed with epilepsy/seizure **Exceptions to the Exclusion Criteria are allowed in the following circumstances: Certain implants, tattoos, metallic objects, or jewelry may be allowed and can be considered safe to image after having been reviewed and approved of by the Purdue MRI Staff. If it is safe, you will be able to participate in the study.

• Dennis A Savaiano, Doctoral, Principal investigator, Purdue University