Identification of Physiological Biomarkers of Gastro-intestinal Discomforts Induced by Milk Consumption
Condition: Healthy Subjects
Study type: Observational

Summary: 
      This study aims to explore digestibility of lactose and milk proteins, gut permeability,
      microbiota composition and psychological wellbeing status in healthy subjects who are
      non-habitual milk consumers due to milk-related gastro-intestinal discomfort in comparison
      with healthy and habitual milk consumers.
    

Detailed description: 
      Individuals reporting digestive disorders associated to milk and dairy products consumption
      increase worldwide, but in many cases this condition is not associated with a well documented
      diagnosis of lactose intolerance or casein allergy. Some investigators hypothesize that these
      disorders (in the absence of any diagnosis of disease) may be due to certain peptides forming
      during milk digestion. Further evidence support the hypothesis that other factors, such as a
      lacking activity of intestinal brush border enzymes, an altered gut permeability, and/or a
      specific gut microbiota composition, may be involved in the onset of gastrointestinal (GI)
      discomforts induced by milk consumption. Indeed, all these factors could modulate the amount
      and type of peptides and lactose skipping digestion and passing through the intestinal
      mucosa, leading to effects on GI motility and gastro-intestinal hormone secretion. Similarly,
      due to its implications in homeostatic and non-homeostatic regulation of eating behaviour as
      well as in pain-regulating processes, a role of endocannabinoid system (ECs) in the evolution
      of GI symptoms post-milk consumption could be hypothesized.

      In this study, the digestibility of lactose and milk proteins will be explored in healthy
      subjects who are non-habitual milk consumers (NHMC) because of gastro-intestinal discomforts
      induced by milk ingestion, in comparison with healthy and habitual milk consumers (HMC). The
      gut permeability, the composition of intestinal microbiota and the psychological wellbeing
      status of subjects will be also investigated and compared in the 2 groups.

      The study design, after the enrollment of participants based on pre-recruitment
      questionnaires and a lactose breath test, includes a gut permeability test and a milk test.

      The potential eligibility of subjects to participate in this study will be assessed through
      pre-recruitment questionnaires, collecting personal and socio-demographic data of volunteers,
      information about milk consumption habits and associated symptoms, general information about
      health (anthropometry, health status, smoking and alcohol consumption habits), household
      environment, information on volunteer's consideration about milk product and its consumption,
      and food habits.

      Potential eligible subjects who will sign the informed consent form, will be assessed for
      final eligibility by mean of a lactose breath test, performed according to the protocol
      approved during the Rome Consensus Conference (2009). GI symptoms will be also recorded by
      subjects, in parallel with H2 measurements in breath samples and up to 24 hours from the
      beginning of the test, by mean of Visual Analogue Scales (VAS).

      During the lactose breath test, subjects will be asked to fill out the physical activity
      questionnaire (short version of the IPAQ), questionnaires on quality of life, on depression,
      anxiety and stress (DASS), a semi-quantitative food frequency questionnaire (FFQ), the King's
      Stool Chart, to evaluate frequency and consistency of feces, and the Three Factor Eating
      Questionnaire (TFEQ) to evaluate individual eating behaviour. Anthropometric data of weight
      and height will be measured as well.

      The overall symptoms evolved during and after the lactose breath test will be assessed
      together with the H2 concentrations measured in breath samples; a cut-off value of 20 ppm
      over the baseline will be used to identify lactose malabsorbers.

      This selection procedure will identify as eligible:

        -  subjects who are negative to breath test (BT-) and do not refer any GI symptom (lactose
           absorber) or refer some minor gastro-intestinal symptoms

        -  subjects who are positive to breath test (BT+) but without any symptom (lactose
           malabsorbers without symptoms).

      On the other hand, subjects will be excluded from the study if:

        -  they are positive to breath test (BT+) and report GI symptoms (lactose intolerant)

        -  they are negative to breath test (BT-) but report severe GI symptoms such as "vomiting"
           and "Loose, mushy or watery stools".

      The enrollment will be concluded when 2 groups of subjects will be constituted as follow

        -  Group 1, habitual milk consumers (HMC): 20 subjects who are regular milk consumers (>700
           mL/week), do not experience GI discomfort after milk intake and are lactose tolerant or
           lactose malabsorber.

        -  Group 2, non-habitual milk consumers (NHMC): 20 subjects who are not regular milk
           consumers (< 150 mL/week), because of GI discomforts after milk intake and are lactose
           tolerant or lactose malabsorber.

      For all subjects from both groups the gut permeability test and the milk test will be
      performed. These tests will be scheduled after one week from the lactose breath test and with
      one week in between. Subjects will be also asked to collect a fecal sample under usual
      dietary habits.

      For gut permeability test, subjects will receive instruction about drug consumption and will
      be asked to follow a controlled diet for 2 days before the test (avoiding milk and dairy
      products, and food products containing polyols).

      Fasting subjects will collect a baseline urine sample, before drinking a solution containing
      5 g lactulose, 2 g mannitol and 2 g sucralose in 100 ml of water. Urines will be collected
      for time periods 0-5 h and 5-24 h in two containers added with 1 mL of a chlorhexidine
      solution at 1 ppm. Urine samples collected over 24 h and delivered the day after to the study
      centre will be aliquoted (1.5 ml) and frozen at -20°C until analyses.

      During the week before the milk test, subjects will fill a food diary. Moreover they will be
      instructed to not drink any milk, dairy product and any food product containing milk proteins
      during the 2 days before the milk test. On the day of milk test, fasting subjects will fill
      out questionnaires about their actual appetite and GI symptoms, baseline urine and blood
      samples will be collected, and fasting glycaemia by finger-prick will be measured.
      Subsequently, subjects will drink 250 ml of milk within 10 min and thereafter, glycaemia,
      symptoms and appetite questionnaires, as well as blood samples and urine will be collected at
      specific time points. After the last blood drawing subjects will be offered a lunch and,
      before leaving the study centre, they will be instructed to fill out GI symptoms and appetite
      questionnaires, to collect urine, to fill out a food diary, and to consume a fixed dinner.
      Questionnaires, diaries and 6-24h urine samples will be delivered to the laboratory on the
      next day.

      Blood samples, stored at -40°C will be analyzed for:

        -  gastro-intestinal hormones (insulin, gastric inhibitory peptide (GIP), glucagon-like
           peptide 1 (GLP-1), glucagon, c-peptide, ghrelin, leptin) in plasma samples pre-treated
           with aprotinin;

        -  endocannabinoids and N-acyl-ethanolamines (ECs);

        -  casein digestion-derived peptides (beta-casomorphins and all known peptides derived from
           milk proteins);

        -  aminoacid profile;

        -  DPPIV activity;

        -  baseline DPPIV concentration.

      Urine samples, aliquoted (1.5 ml) and frozen at -40°C, will be used to measure:

        -  lactose, galactose + glucose, and indican excretion levels (for urines collected over 24
           h after milk test).

        -  lactulose, mannitol and sucralose excretion levels (for urines collected over 24 h after
           gut permeability test).

      Fecal samples, stored at -40°C, will be used for DNA extraction according to the standard
      operating procedures of the International Human Microbiome Consortium
      (http://www.human-microbiome.org/). Statistical analysis will be carried out by the most
      appropriate methods in order to infer the associations between specific microbiota signatures
      and clinical, dietary and metabolomic parameters.
    

Intervention name(s): Lactose Breath Test; Gut permeability test; Milk Test
Intervention type(s): Diagnostic Test; Other; Other
Intervention description(s): Lactose Breath Test is performed during the recruitment step. Fasting subjects are asked to ingest 20 g of lactose in a 10% water solution. Breath samples are collected at baseline and every 30 min after lactose ingestion, for an overall test duration of 4 h. A cut-off value of 20 ppm over the baseline will be used to identify lactose malabsorbers.; A 3-sugar probes test is conducted by administering 5 g lactulose, 2 g mannitol and 2 g sucralose in water solution to fasting subjects. Urine are collected at baseline (before sugar ingestion) and for time periods 0-5 h and 5-24 h.; Milk test will be performed by administering to fasting subjects a portion of 250 ml of semi-skimmed milk treated with ultra-high temperature (UHT). Glycaemia measurements, blood withdrawals and questionnaires completion will be performed at baseline and repeated after 30 minutes, 1, 2, 4 and 6 hours after milk ingestion. Urine collection will be performed at baseline and for 6 time intervals after milk ingestion (0-2, 2-4, 4-6, 6-8, 8-12, 12-24 hours).
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Community sample. Recruitment will be done through public announcements in the department
        places, local ambulatories and shops, and invitation through social networks.
      
Biospecimen description: 
      serum, plasma, plasma pre-treated with aprotinin, urine, feces
    
Keywords: milk digestibility; gastro-intestinal discomfort; milk proteins; lactose; gut permeability; gut microbiota