Identification of Physiological Biomarkers of Gastro-intestinal Discomforts Induced by Milk Consumption
Conditions: Healthy Subjects
Sponsor: Paola Vitaglione

 
Study Protocol 
with 
Statistical Analysis Plan 
 
Identification of Physiological Biomarkers of Gastro-intestinal 
Discomforts Induced by Milk Consumption 
 
MIDI 
 
 
NCT04205045 
 
 
Version: 1 
Date: 26/07/2022 
 
 
Author: Paola Vitaglione, University of Naples, Federico II 
 
 
Principal Investigator: Paola Vitaglione, University of Naples, Federico II 
 
 
 
 
 
 
 
 
 
 
 

Background and rationale 
Gastro-intestinal discomforts associated with the consumption of milk and dairy products are frequent 
in the general population, even in absence of lactose intolerance and casein allergy. Evidence from 
the literature suggests that these disorders (in the absence of other diagnoses) may be due to some 
peptides produced during the digestion of milk proteins. Moreover, some scientific evidence suggests 
that other factors, such as an altered intestinal permeability and/or a specific composition of the 
intestinal microbiota, may be responsible for gastrointestinal (GI) disorders resulting from milk 
consumption. It is also possible to hypothesize a role of the endocannabinoid system in the evolution 
of GI symptoms after milk ingestion given its implications in homeostatic and non-homeostatic 
regulation of feeding behavior, as well as in pain regulation processes. 
Objectives  
 
This study aims to compare two groups of healthy (non-lactose intolerant) subjects who are irregular 
milk consumers, due to milk induced gastrointestinal disorders, with another group of healthy subjects 
who consume milk regularly, without symptoms, in relation to the primary and secondary objectives 
here described. 
 
By performing a milk test in which volunteers ingest 250 ml of UHT semi-skimmed milk, primary 
objectives include the evaluation of: 
● plasmatic response of milk-derived peptides; 
● plasmatic profile of GI hormones; 
● plasmatic profile of endocannabinoids and N-acyl-ethanolamines.   
● urinary profile of lactose over 24 h;  
 
Secondary objectives include the assessment of: 
● baseline and postprandial plasma profile of aminoacids; 
● baseline and postprandial blood glucose; 
● baseline and postprandial GI symptoms;  
● baseline serum concentration of dipeptidyl peptidase IV (DPPIV); 
● baseline and postprandial serum activity of dipeptidyl peptidase IV (DPPIV); 
● urinary excretion of indican as marker of protein digestibility; 
● correlations of GI symptoms with appetite ratings and blood glucose, plasma GI hormones, 
plasma peptides and amino acids as well as with urinary excretion of lactose and galactose + 
glucose;  
● the composition of the intestinal microbiota (metagenomic analysis); 
● the psychological profile through questionnaires on the perception of quality of life, on levels 
of depression, anxiety and stress and on eating behavior. 
 
Further secondary objectives include the assessment of: 
● the prevalence of lactose malabsorption in the two groups, by using results from Lactose H2 
breath test performed during the recruitment procedure; 
● urinary excretion of lactulose, mannitol and sucralose as markers of intestinal permeability 
after an oral challenge of these 3 sugar probes. 
 
 
 
 
 

 
Trial design 
Recruitment  
The recruitment process is based on: 
● Pre-recruitment questionnaires to collect information on dietary habits and milk-related  
symptoms; 
● Lactose breath test for the assessment of final eligibility. After lactose ingestion, H2 
measurements in breath will be performed over 4 hours; a cut-off value of 20 ppm over the 
baseline will be used to identify lactose malabsorbers. In parallel, and up to 24 hours, subjects 
will also record GI symptoms, through Visual Analogue Scales (VAS). 
The eligibility of volunteers to participate in the study is defined on the basis of the following 
inclusion and exclusion criteria: 
Inclusion Criteria: 
● Drinking milk (maximum 150 ml/week for non-habitual milk consumers and minimum of 
700 ml/week for habitual milk consumers). 
● BMI between 18.5 - 30 kg/m2. 
● Availability to participate into three visits (with one week in between). 
● Willing to drink 250 ml of milk in fasting condition within 10 min on one occasion. 
● Signature of a written informed consent. 
● Negative lactose breath test result (increase in H2 concentrations < 20 ppm vs basal value) 
and without symptoms or with any symptoms except "vomiting" and "Loose, mushy or 
watery stools". 
● Lactose malabsorbers (increase in H2 concentrations > 20 ppm vs basal value but without 
symptoms). 
Exclusion Criteria: 
● Presence of any relevant organic, systemic or metabolic disease or abnormal laboratory 
values. 
● Ascertained intestinal organic diseases, including celiac disease or inflammatory bowel 
diseases. 
● Previous major abdominal surgeries. 
● Active malignancy of any type, or history of a malignancy (patients with a history of other 
malignancies that have been surgically removed and who have no evidence of recurrence for 
at least five years before study enrolment are also acceptable). 
● Untreated food intolerance. 
● Lactose intolerant 
● Assumption of probiotics or topic and/or systemic antibiotic therapy during the last month. 
● Systematic/frequent assumption of contact laxatives. 
● Pregnant and lactating women. 
● Inability to conform with protocol. 
● Treatment with any investigational drug within the previous 30 days. 

● Recent history or suspicion of alcohol abuse or drug addiction. 
● Subjects having symptoms of "vomiting" and "Loose, mushy or watery stools" at any level 
of severity following milk consumption and the lactose breath test 
Allocation 
Subjects who will be assessed as eligible and will accept to enter the study protocol, will be allocated 
in the proper group, and the enrollment will be concluded when 2 groups of subjects will be 
constituted as follows: 
● Group 1, habitual milk consumers (HMC): 20 subjects who are regular milk consumers (>700 
mL/week), do not experience GI discomforts 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. 
Intervention and other measures/samples 
The study protocol includes a gut permeability test and a milk test to be performed for all subjects 
from both groups after one week from the lactose breath test and with one week in between.  
Gut permeability test 
Subjects will receive instruction to avoid medicine consumption and to follow a controlled diet for 2 
days before the test (avoiding milk and dairy products, and food products containing polyols that 
were included in a list of products they received). 
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 hours and 5-24 hours in two containers containing 1 mL of a chlorhexidine solution at 1 ppm.  
Urine samples collected over 24 hours and delivered the day after to the study centre will be aliquoted 
(1.5 ml) and frozen at −40°C until analyses that include lactulose, mannitol and sucralose 
concentration and baseline creatinine concentration. 
The ratio between lactulose and mannitol (L / M) in the urine collected within the first 5 hours will 
represent permeability in the small intestine, while the total amount of sucralose excreted in urine in 
the 24 hours will be used to evaluate the colonic permeability. 
Milk test 
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 the 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 (time 0). 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 (time 0.5, 1, 2, 4 and 6 hours after milk 
ingestion). After the last blood drawing, subjects will be offered 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 1-day 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 will be centrifuged at 4°C and 4000 rpm; serum, plasma and plasma + aprotinin 
samples will be aliquoted and stored at −40°C. 
Plasma samples will be analyzed for:  
● casein digestion-derived peptides (beta-casomorphins and all known peptides derived from 
milk proteins); 
● aminoacid profile;  
● endocannabinoids and N-acyl-ethanolamines (ECs). 
Plasma samples added with aprotinin will be analyzed for:  
● gastro-intestinal hormones (in plasma samples pre-treated with aprotinin): insulin, GIP, GLP-
1, glucagon, c-peptide, ghrelin, leptin. 
Serum samples will be analyzed for: 
● DPPIV activity; 
● baseline DPPIV concentration. 
In urine samples, aliquoted (1.5 ml) and frozen at −40°C, all the following analytes will be measured: 
● lactose excretion levels; 
● galactose + glucose excretion levels; 
● indican excretion levels; 
● baseline creatinine concentration. 
Subjects will be also asked to collect a fecal sample under usual dietary habits and to fill out 
questionnaires as follows: 
● physical activity questionnaire (short version of the IPAQ); 
● questionnaire on quality of life; 
● questionnaire on depression, anxiety and stress (DASS); 
● semi-quantitative food frequency questionnaire (FFQ); 
● the King's Stool Chart, to evaluate frequency and consistency of feces; 
● Three Factor Eating Questionnaire (TFEQ) to evaluate individual eating behaviour. 
Anthropometric data of weight and height will be measured as well.  
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 
variables. 
 
 
 
 

Statistical analysis plan 
 
Sample size calculation 
 
The sample size for this study has been calculated on the basis of the primary endpoints here listed: 
● milk-derived peptides plasma concentrations  after milk consumption; 
● lactose urinary excretion after milk consumption; 
● post-prandial plasma profile of GI hormones and ECs. 
 
Considering circulating mean values and standard deviations reported by Deth et al. (2016) in blood 
of healthy population, power analysis indicated that a sample size of 19 participants per group is 
sufficient to detect a minimum variation of 40% in circulating BCM-7 with a power of 80% and an α 
= 0.05.  
Considering excreted % of lactose in urine, hydrogen concentration in breath and relative standard 
deviations as reported by Bezerra et al. (1990) a sample size of 19 participants per group is sufficient 
to detect a 15% minimum variation of lactose and a 50% minimum variation of hydrogen in healthy 
subjects, with a power of 80% and an α = 0.05. 
Regarding post-prandial plasma profile of GI hormones and ECs, our previous studies showed that a 
sample size of 13 participants is adequate to find significant differences with an 80% power and an α 
= 0.05. (Mennella et al., 2015; Mennella et al., 2016).  
 
The participant number will be increased up to 40 (20 subjects per group) in view of possible 
dropouts. 
 
Statistical analysis 
 
Data will be expressed as means ± SEMs, unless otherwise specified.  
Two-tailed P values lower than 0.05 were considered significantly different 
Missing data will be handled as such, i.e., no imputation of missing data will be performed. 
Categorical variables 
For variables with 2 categories, Chi-square test will be used to evaluate the association between 
variables if the expected values in each cell are ≥ 5 in at least 25% of the cells. When the expected 
values are < 5 in more than 25 % of the cells, Fisher’s exact test will be used. 
To assess the association between two polytomous variables (>2 categories), the Pearson’s Chi-
square test will be used if the frequency expected is ≥ 5 in at least 75% of the cells. If not, we will 
regroup to increase the expected frequencies. 
Continuous variables 
Normality of continuous variables will be assessed using the Shapiro-Wilk test. For non-normal 
variables showing a significant positive skewness, logarithmic, square-root, exponential, or inverse 
transformation will be applied. For those variables which will not show a normal distribution after 
arithmetic transformation, non-parametric tests will be used. 
 
 
 
 

Continuous variables without repeated measures 
For those variables which will show a normal distribution at the Shapiro-Wilk test,  an independent 
two-sample T test will be performed to check differences between groups, whereas ANCOVA 
analysis will be performed to include covariates in the analysis. 
 
Differences between groups of non-normally distributed variables will be assessed by Mann-Whitney 
U test, while non-parametric analysis of covariance (Quade’s test) will be used to explore the effects 
of any covariates. 
 
Continuous variables with repeated measures 
Normality of measures, for each time, will be assessed using the Shapiro-Wilk test. 
Differences of variables between baseline and over time within and between the groups, for normally 
distributed variables, will be tested by ANOVA with repeated measures on one factor (time) and 
Bonferroni adjustment for multiple comparisons. For non-normally distributed variables, Friedman 
test and pairwise Wilcoxon as a post hoc test will be applied.  
Analysis of quartiles will be performed for both baseline values and pairwise time points (∆) upon 
milk ingestion. 
For these variables with repeated measures, the total area under the curves (AUCs) will be also 
estimated using the linear trapezoidal rule, and differences in AUCs between the groups will be 
assessed by proper parametric or non-parametric analyses including as covariates the potential 
confounding variables measured at baseline. 
 
Pearson’s correlation coefficients will be calculated to assess bivariate associations among 
continuous variables at baseline or partial associations of estimated AUCs controlling for baseline 
values for variables with repeated measures. 
Spearman’s rank correlation will be used to assess association between non-parametric variables 
collected and microbiota related variables (e. g. species abundance and genes abundance). 
In order to explore differences in microbiome profiles, a Partial Least Squares Discriminant Analysis 
was applied. 
Furthermore, principal component analyses (PCA) on specific variables at baseline or unstandardised 
residuals correlation between AUCs and related baseline values will be also performed in order to 
extract features relevant to the cluster structure. Moreover, statistical significance of the distance 
between groups in the PCA analysis will be computed using the Hotelling T2 test.  
Further analyses 
Since the prevalence for lactose malabsorption over the enrollment will be assessed, all the statistical 
analyses reported above may be applied to subgroups of subjects classified as “lactose tolerants (LT)” 
and “lactose malabsorbers (LM)” in order to check differences between the two groups and/or 
correlations between variables assessed. 
Chi-means will allow to assess differences between subgroups classified on the basis of lactose 
absorption capacity of subjects as detected by lactose H2 breath test. 
Statistical analyses and data visualization will be carried out in R environment (http://www.r-
project.org).