xml: <clinical_study>
    <!-- This xml conforms to an XML Schema at:
      https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
    <required_header>
      <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
      <link_text>Link to the current ClinicalTrials.gov record.</link_text>
      <url>https://clinicaltrials.gov/ct2/show/NCT06028659</url>
    </required_header>
    <id_info>
      <org_study_id>340/2023/SPER/UNIPR</org_study_id>
      <nct_id>NCT06028659</nct_id>
    </id_info>
    <brief_title>Investigation of in Vivo Endogenous and/or Exogenous Production of Phenolic Metabolites Using (un)Targeted Metabolomics</brief_title>
    <acronym>ENDOPHENOL</acronym>
    <official_title>Investigation of in Vivo Endogenous and/or Exogenous Production of Phenolic Metabolites Using (un)Targeted Metabolomics</official_title>
    <sponsors>
      <lead_sponsor>
        <agency>University of Parma</agency>
        <agency_class>Other</agency_class>
      </lead_sponsor>
    </sponsors>
    <source>University of Parma</source>
    <oversight_info>
      <is_fda_regulated_drug>No</is_fda_regulated_drug>
      <is_fda_regulated_device>No</is_fda_regulated_device>
    </oversight_info>
    <brief_summary>
      <textblock>
        Plant (poly)phenols is what we call a large number of substances that are produced by plants&#xD;
        as secondary plant metabolites, which means substances that are not used for their growth and&#xD;
        development but are necessary for them to survive. (Poly)phenols are divided in two major&#xD;
        groups, flavonoids, and non-flavonoids, and each group contains a varied set of subgroups and&#xD;
        substances. They are widely spread in fruit and vegetables that are part of the human diet,&#xD;
        and, in general, studies have attributed many biological effects to the ingestion of&#xD;
        (poly)phenols, especially in the prevention of non-communicable diseases. For this reason,&#xD;
        research aims to understand their role in the health benefits of a diet that is rich in&#xD;
        fruits and vegetables. When ingested, (poly)phenols are digested by both the human organism&#xD;
        and the gut microbiota and are broken down into several smaller substances (catabolites) that&#xD;
        are called low-molecular weight (poly)phenols (LMWP). Most of the absorbed (poly)phenols that&#xD;
        reach our bloodstream and organs are LMWP.&#xD;
  &#xD;
        For the proposed study, 30 healthy adults will be recruited and, if considered able to&#xD;
        participate, will follow a standardized diet that is restricted in (poly)phenol intake and&#xD;
        will be randomly divided into two groups: one will receive a known source of (poly)phenols&#xD;
        (coffee) and the other will receive water, keeping the restriction of (poly)phenol from the&#xD;
        diet. The duration of one phase is 4 days + 12 hours, during which urine, feces, and saliva&#xD;
        will be collected. Then, after a 2-week-interval, subjects will repeat the experiment, except&#xD;
        that this time the group who had coffee will have water, and vice versa. Again, urine and&#xD;
        feces will be collected. The objective of the study is to identify and quantify LMWP mainly&#xD;
        in urine, but also in feces, and try to understand how much was produced when there was no&#xD;
        (poly)phenols in the diet compared with when there was ingestion of coffee (poly)phenols. The&#xD;
        production of LMWP without coffee could be because of their production from other sources,&#xD;
        like the metabolism of amino acids, proteins, and catecholamines (i.e. dopamine). The&#xD;
        composition of the gut microbiota and relevant genetic information can alter the metabolism&#xD;
        of (poly)phenols and will be considered in the analyses. Knowing how much of LMWP actually&#xD;
        comes from the diet is important to understand the relevance and health benefits of these&#xD;
        molecules.&#xD;
      </textblock>
    </brief_summary>
    <detailed_description>
      <textblock>
        The study will consist of a randomized cross-over trial and each arm will last 5 days. A&#xD;
        total of thirty (30) healthy volunteers will be recruited for the study in Parma (PR), using&#xD;
        announcements placed in the university, hospital, and public places. To allocate subjects to&#xD;
        each arm, a list will be generated using a randomized block design by using Random Number&#xD;
        Generator Pro (Segobit Software). This list will be blind for the PI and volunteers, as it&#xD;
        will be made by a colleague not involved in the subjects' enrolment and using a numbered&#xD;
        sequence in sealed, opaque envelopes.&#xD;
  &#xD;
        Volunteers freely accepting to participate to the study will be asked for their written&#xD;
        informed consent. Information to the volunteers will be provided before and separately from&#xD;
        the consent form. These information sheets and the informed consent forms have been prepared&#xD;
        according to the Oviedo Convention and consider the guide of the local Ethics Committee.&#xD;
        After obtaining their written informed consent, volunteers will undergo a screening visit to&#xD;
        determine eligibility and collect personal data.&#xD;
  &#xD;
        Privacy of the volunteers will be assured through pseudoanonymization by the attribution of a&#xD;
        personal alphanumeric code. Recruited volunteers will also sign an authorisation to the use&#xD;
        of personal information and retrieved data.&#xD;
  &#xD;
        After recruitment, subjects will attend a first visit, when personal data will be collected,&#xD;
        and body composition and anthropometry parameters measured. Then, they will receive the&#xD;
        foods, supplements, and materials necessary to follow the study. The recruited subjects will&#xD;
        be requested to follow a low-(poly)phenol diet, which will be the same for all the volunteers&#xD;
        and maintained for the whole period of the intervention study. The foods, preparation mode,&#xD;
        and times for eating the meals will be provided by the sponsor. The diet will be personalised&#xD;
        to approximate the necessary calories based on the measured basal metabolic rate, multiplied&#xD;
        by the physical activity level set at 1.45, and there will be quantitative variations on the&#xD;
        low-(poly)phenol diet based on the daily energy requirement: 1500 kcal/day, 1750 kcal/day,&#xD;
        2000 kcal/day, 2250 kcal/day, 2500 kcal/day, 2750 kcal/day, or 3000 kcal/day, all with&#xD;
        nutritionally balanced amounts of protein, fat, and carbohydrates. In addition, participants&#xD;
        will be requested to restrict physical exercise during the study duration.&#xD;
  &#xD;
        Subjects will be asked to collect urine produced during the 60h prior the intervention,&#xD;
        starting from the evening prior the first day of diet (from 21.00 of the night prior the&#xD;
        first day of diet) and every 12 hours (60-48h, 48-36h, 36-24h, 24-12h, 12-0h), and to&#xD;
        complete the food diary to check for compliance to the diet. Subjects are asked to bring&#xD;
        collected urine to the the Human Nutrition Unit of the Food and Drug Department of the&#xD;
        University of Parma.&#xD;
  &#xD;
        On the morning of the third day of diet, subjects will be requested to arrive at Parma&#xD;
        University in fasting conditions. Prior to receiving the intervention, fasting spot urine and&#xD;
        saliva samples will be collected. Then, sugared-decaffeinated-coffee (3 doses of 50 mL each&#xD;
        and 5g of sugar) or sugared-water (150 mL of water and 5g of sugar) will be provided&#xD;
        according to randomization of the arms, along with a standardized breakfast consisting of a&#xD;
        (poly)phenol-free, nutritionally balanced solid meal and water (ad libitum).&#xD;
  &#xD;
        After breakfast, subjects will be asked to autonomously collect urine for the next 48h at&#xD;
        specified timepoints (0 - 3h; 3 - 6h; 6 - 9h; 9 - 12h; 12 - 24h; 24 - 36h; and 36 - 48h),&#xD;
        following the same personalized diet. Lastly, subjects will autonomously collect a fecal&#xD;
        sample within 6 to 48h after the intervention.&#xD;
  &#xD;
        On the morning of the last day, after the last urine collection, subjects will bring&#xD;
        collected urine and fecal samples and the 5-day food diary to the Human Nutrition Unit of the&#xD;
        Food and Drug Department of the University of Parma.&#xD;
  &#xD;
        After the first arm, subjects will resume their habitual lifestyles for 2 consecutive weeks,&#xD;
        subsequently, they will attend another visit, when all the anthropometric evaluations carried&#xD;
        out during the first visit will be repeated and another kit with foods and information for&#xD;
        the personalized diet will be given. With the study being a cross-over design, the protocol&#xD;
        followed will be the same as the one described above (except for saliva sampling, which will&#xD;
        only be performed once). Coming the third day of diet, subjects who ingested coffee in the&#xD;
        prior arm of the study will ingest water, and vice-versa.&#xD;
  &#xD;
        For this study, the sample size was estimated considering the mean concentration of&#xD;
        coffee-derived (poly)phenols and their metabolites in urine as the primary outcome and the&#xD;
        available literature on similar approaches and studies of the area. The number of 30&#xD;
        volunteers was established considering the accepted parameters for clinical trials (power set&#xD;
        at 0.8 and α set at 0.05) possible dropouts (15% drop-out rate), and exclusion of volunteers&#xD;
        to maintain the sample power for statistical analysis. Before any comparison is performed,&#xD;
        the normality of each variable will be evaluated to choose the most appropriate statistical&#xD;
        test. If metabolite data are normally distributed, data will be expressed as mean ± standard&#xD;
        deviation and analysed using repeated measures t-test or ANOVA with post-hoc comparisons. If&#xD;
        data are not normally distributed, they will be reported as median and Interquartile Range&#xD;
        and the Friedman test with post hoc pairwise comparisons will be performed. P-value &lt; 0.05&#xD;
        will be regarded as statistically significant.&#xD;
  &#xD;
        Unsupervised and supervised multivariate analysis will be used to identify and characterize&#xD;
        the different urinary metabotypes, following the procedures developed by our research group&#xD;
        for targeted metabolomics analysis of phenolic metabolites or when using untargeted&#xD;
        metabolomics data. We will be using different clustering approaches to better explore the&#xD;
        existing variability. This will serve to establish the number of clusters (i.e., phenolic&#xD;
        metabotypes), the number of participants forming each cluster and the similarities among&#xD;
        possible metabotypes.&#xD;
  &#xD;
        The relationship between patterns of LMWP and their putative determinants (age, sex,&#xD;
        ethnicity, lifestyle, anthropometrics, body composition, genotype, and gut microbiome) will&#xD;
        be explored using correlation tests and multivariate analyses. P-value &lt; 0.05 will be&#xD;
        regarded as statistically significant. Statistics will be carried out by the specialized&#xD;
        staff of the Human Nutrition Unit, under the supervision of the PI, and in collaboration with&#xD;
        the University teams in charge of microbial profiling and genotyping.&#xD;
      </textblock>
    </detailed_description>
    <overall_status>Not yet recruiting</overall_status>
    <start_date type="Anticipated">October 2023</start_date>
    <completion_date type="Anticipated">February 2024</completion_date>
    <primary_completion_date type="Anticipated">December 2023</primary_completion_date>
    <phase>N/A</phase>
    <study_type>Interventional</study_type>
    <has_expanded_access>No</has_expanded_access>
    <study_design_info>
      <allocation>Randomized</allocation>
      <intervention_model>Crossover Assignment</intervention_model>
      <primary_purpose>Screening</primary_purpose>
      <masking>Triple (Participant, Investigator, Outcomes Assessor)</masking>
    </study_design_info>
    <primary_outcome>
      <measure>Number and concentration of phenolic metabolites in urine</measure>
      <time_frame>Samples acquired from 60 hours prior to 48 hours after intervention or control</time_frame>
      <description>Urine samples will be subjected to both targeted and untargeted liquid chromatography mass spectrometry analyses</description>
    </primary_outcome>
    <secondary_outcome>
      <measure>Identification and quantification of gut bacteria (genera and species) in feces</measure>
      <time_frame>Samples acquired from 6-48 hours after coffee or control</time_frame>
      <description>Analysis of microbiota taxonomy at species level from bacterial DNA extracted from fecal samples.</description>
    </secondary_outcome>
    <secondary_outcome>
      <measure>Number and concentration of phenolic metabolites in feces</measure>
      <time_frame>Samples acquired from 6-48 hours after coffee or control</time_frame>
      <description>Fecal samples will be subjected to both targeted and untargeted liquid chromatography mass spectrometry analyses</description>
    </secondary_outcome>
    <secondary_outcome>
      <measure>Identification and quantification of metabolites after in vitro fecal fermentation</measure>
      <time_frame>Samples acquired from 6-48 hours after coffee or control</time_frame>
      <description>A set of (poly)phenol-rich substrates and protein-rich food items will be incubated and fermented in sealed jars to evaluate the ability of human microbiota to produce different phenolic metabolites.</description>
    </secondary_outcome>
    <secondary_outcome>
      <measure>Identification of single-nucleotide polymorphism analysis</measure>
      <time_frame>Single saliva samples collected from 1 hour to 30 minutes before the intervention with coffee or control.</time_frame>
      <description>Genotyping for possibile genetic variations in genes related to (poly)phenol kinetics. Commercially available SNP arrays detect gene variability across the majority of the genome using a tagging SNP approach.</description>
    </secondary_outcome>
    <number_of_arms>2</number_of_arms>
    <enrollment type="Anticipated">30</enrollment>
    <condition>Individual Variability in (Poly)Phenol Metabolism</condition>
    <arm_group>
      <arm_group_label>Coffee</arm_group_label>
      <arm_group_type>Experimental</arm_group_type>
      <description>In this arm, volunteers will receive, once, 3 decaffeinated coffees (150 mL total) with sugar as a known source of (poly)phenols.</description>
    </arm_group>
    <arm_group>
      <arm_group_label>Control</arm_group_label>
      <arm_group_type>Other</arm_group_type>
      <description>In this arm, volunteers will receive, once, 150 mL of sugared water as control.</description>
    </arm_group>
    <intervention>
      <intervention_type>Other</intervention_type>
      <intervention_name>Coffee</intervention_name>
      <description>Decaffeinated coffee with 1 pack (5g) of sugar from commercially available brands prepared with commercially available domestic coffee machines.</description>
      <arm_group_label>Coffee</arm_group_label>
    </intervention>
    <intervention>
      <intervention_type>Other</intervention_type>
      <intervention_name>Water</intervention_name>
      <description>150 mL of hot water with 1 pack (5g) of sugar from commercially available brands prepared with commercially available domestic coffee machines.</description>
      <arm_group_label>Control</arm_group_label>
    </intervention>
    <eligibility>
      <criteria>
        <textblock>
          Inclusion Criteria:&#xD;
  &#xD;
            -  Apparently healthy adults,&#xD;
  &#xD;
            -  Age 20-40 years old,&#xD;
  &#xD;
            -  Body mass index (BMI) between 18-28,&#xD;
  &#xD;
            -  Regular coffee consumers (at least 1 cup a day).&#xD;
  &#xD;
          Exclusion Criteria:&#xD;
  &#xD;
            -  Clinical diagnosis of metabolic diseases,&#xD;
  &#xD;
            -  Clinical diagnosis of cardiovascular diseases,&#xD;
  &#xD;
            -  Clinical diagnosis of renal diseases,&#xD;
  &#xD;
            -  Clinical diagnosis of digestive diseases,&#xD;
  &#xD;
            -  Immunodeficiency&#xD;
  &#xD;
            -  Autoimmune diseases (other than well-compensated hypothyroidism),&#xD;
  &#xD;
            -  Mental disorders&#xD;
  &#xD;
            -  Regular use of medication (except birth-control pills and hormone replacement&#xD;
               therapies),&#xD;
  &#xD;
            -  Use of antibiotics in the last month prior to enrollment,&#xD;
  &#xD;
            -  Food allergies or intolerances,&#xD;
  &#xD;
            -  Pregnancy&#xD;
  &#xD;
            -  Lactation&#xD;
        </textblock>
      </criteria>
      <gender>All</gender>
      <minimum_age>20 Years</minimum_age>
      <maximum_age>40 Years</maximum_age>
      <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
    </eligibility>
    <overall_official>
      <last_name>Pedro M Mena Parreño, PhD</last_name>
      <role>Principal Investigator</role>
      <affiliation>University of Parma</affiliation>
    </overall_official>
    <overall_contact>
      <last_name>Pedro M Mena Parreño, PhD</last_name>
      <phone>+393288087332</phone>
      <email>pedromiguel.menaparreno@unipr.it</email>
    </overall_contact>
    <overall_contact_backup>
      <last_name>Letizia Bresciani, PhD</last_name>
      <email>letizia.bresciani@unipr.it</email>
    </overall_contact_backup>
    <location>
      <facility>
        <name>University of Parma - Plesso Biotecnologico Integrato</name>
        <address>
          <city>Parma</city>
          <state>PR</state>
          <zip>43125</zip>
          <country>Italy</country>
        </address>
      </facility>
    </location>
    <location_countries>
      <country>Italy</country>
    </location_countries>
    <verification_date>August 2023</verification_date>
    <study_first_submitted>August 23, 2023</study_first_submitted>
    <study_first_submitted_qc>August 31, 2023</study_first_submitted_qc>
    <study_first_posted type="Actual">September 8, 2023</study_first_posted>
    <last_update_submitted>August 31, 2023</last_update_submitted>
    <last_update_submitted_qc>August 31, 2023</last_update_submitted_qc>
    <last_update_posted type="Actual">September 8, 2023</last_update_posted>
    <responsible_party>
      <responsible_party_type>Principal Investigator</responsible_party_type>
      <investigator_affiliation>University of Parma</investigator_affiliation>
      <investigator_full_name>Pedro M. Mena Parreño</investigator_full_name>
      <investigator_title>Principal Investigator</investigator_title>
    </responsible_party>
    <keyword>(poly)phenols</keyword>
    <keyword>catabolites</keyword>
    <keyword>coffee</keyword>
    <keyword>metabolism</keyword>
    <keyword>low-molecular-weight (poly)phenols</keyword>
    <keyword>metabolomics</keyword>
    <patient_data>
      <sharing_ipd>Yes</sharing_ipd>
      <ipd_description>The data shared will be those that underlie results in a publication.</ipd_description>
      <ipd_info_type>Study Protocol</ipd_info_type>
      <ipd_info_type>Statistical Analysis Plan (SAP)</ipd_info_type>
      <ipd_time_frame>Data will be available from when published in peer-reviewed journals.</ipd_time_frame>
      <ipd_access_criteria>Data available in publications can be freely accessed. If further data is required to be shared, it will be assessed case by case.</ipd_access_criteria>
    </patient_data>
    <!-- Results have not yet been posted for this study                                          -->
  </clinical_study>
  
  
text: download date: ClinicalTrials.gov processed this data on September 20, 2023
  link text: Link to the current ClinicalTrials.gov record.
  url: https://clinicaltrials.gov/ct2/show/NCT06028659
  org study id: 340/2023/SPER/UNIPR
  nct id: NCT06028659
  lead sponsor: 
  is fda regulated drug: No
  is fda regulated device: No
  Plant (poly)phenols is what we call a large number of substances that are produced by plants
        as secondary plant metabolites, which means substances that are not used for their growth and
        development but are necessary for them to survive. (Poly)phenols are divided in two major
        groups, flavonoids, and non-flavonoids, and each group contains a varied set of subgroups and
        substances. They are widely spread in fruit and vegetables that are part of the human diet,
        and, in general, studies have attributed many biological effects to the ingestion of
        (poly)phenols, especially in the prevention of non-communicable diseases. For this reason,
        research aims to understand their role in the health benefits of a diet that is rich in
        fruits and vegetables. When ingested, (poly)phenols are digested by both the human organism
        and the gut microbiota and are broken down into several smaller substances (catabolites) that
        are called low-molecular weight (poly)phenols (LMWP). Most of the absorbed (poly)phenols that
        reach our bloodstream and organs are LMWP.
  
        For the proposed study, 30 healthy adults will be recruited and, if considered able to
        participate, will follow a standardized diet that is restricted in (poly)phenol intake and
        will be randomly divided into two groups: one will receive a known source of (poly)phenols
        (coffee) and the other will receive water, keeping the restriction of (poly)phenol from the
        diet. The duration of one phase is 4 days + 12 hours, during which urine, feces, and saliva
        will be collected. Then, after a 2-week-interval, subjects will repeat the experiment, except
        that this time the group who had coffee will have water, and vice versa. Again, urine and
        feces will be collected. The objective of the study is to identify and quantify LMWP mainly
        in urine, but also in feces, and try to understand how much was produced when there was no
        (poly)phenols in the diet compared with when there was ingestion of coffee (poly)phenols. The
        production of LMWP without coffee could be because of their production from other sources,
        like the metabolism of amino acids, proteins, and catecholamines (i.e. dopamine). The
        composition of the gut microbiota and relevant genetic information can alter the metabolism
        of (poly)phenols and will be considered in the analyses. Knowing how much of LMWP actually
        comes from the diet is important to understand the relevance and health benefits of these
        molecules.
  The study will consist of a randomized cross-over trial and each arm will last 5 days. A
        total of thirty (30) healthy volunteers will be recruited for the study in Parma (PR), using
        announcements placed in the university, hospital, and public places. To allocate subjects to
        each arm, a list will be generated using a randomized block design by using Random Number
        Generator Pro (Segobit Software). This list will be blind for the PI and volunteers, as it
        will be made by a colleague not involved in the subjects' enrolment and using a numbered
        sequence in sealed, opaque envelopes.
  
        Volunteers freely accepting to participate to the study will be asked for their written
        informed consent. Information to the volunteers will be provided before and separately from
        the consent form. These information sheets and the informed consent forms have been prepared
        according to the Oviedo Convention and consider the guide of the local Ethics Committee.
        After obtaining their written informed consent, volunteers will undergo a screening visit to
        determine eligibility and collect personal data.
  
        Privacy of the volunteers will be assured through pseudoanonymization by the attribution of a
        personal alphanumeric code. Recruited volunteers will also sign an authorisation to the use
        of personal information and retrieved data.
  
        After recruitment, subjects will attend a first visit, when personal data will be collected,
        and body composition and anthropometry parameters measured. Then, they will receive the
        foods, supplements, and materials necessary to follow the study. The recruited subjects will
        be requested to follow a low-(poly)phenol diet, which will be the same for all the volunteers
        and maintained for the whole period of the intervention study. The foods, preparation mode,
        and times for eating the meals will be provided by the sponsor. The diet will be personalised
        to approximate the necessary calories based on the measured basal metabolic rate, multiplied
        by the physical activity level set at 1.45, and there will be quantitative variations on the
        low-(poly)phenol diet based on the daily energy requirement: 1500 kcal/day, 1750 kcal/day,
        2000 kcal/day, 2250 kcal/day, 2500 kcal/day, 2750 kcal/day, or 3000 kcal/day, all with
        nutritionally balanced amounts of protein, fat, and carbohydrates. In addition, participants
        will be requested to restrict physical exercise during the study duration.
  
        Subjects will be asked to collect urine produced during the 60h prior the intervention,
        starting from the evening prior the first day of diet (from 21.00 of the night prior the
        first day of diet) and every 12 hours (60-48h, 48-36h, 36-24h, 24-12h, 12-0h), and to
        complete the food diary to check for compliance to the diet. Subjects are asked to bring
        collected urine to the the Human Nutrition Unit of the Food and Drug Department of the
        University of Parma.
  
        On the morning of the third day of diet, subjects will be requested to arrive at Parma
        University in fasting conditions. Prior to receiving the intervention, fasting spot urine and
        saliva samples will be collected. Then, sugared-decaffeinated-coffee (3 doses of 50 mL each
        and 5g of sugar) or sugared-water (150 mL of water and 5g of sugar) will be provided
        according to randomization of the arms, along with a standardized breakfast consisting of a
        (poly)phenol-free, nutritionally balanced solid meal and water (ad libitum).
  
        After breakfast, subjects will be asked to autonomously collect urine for the next 48h at
        specified timepoints (0 - 3h; 3 - 6h; 6 - 9h; 9 - 12h; 12 - 24h; 24 - 36h; and 36 - 48h),
        following the same personalized diet. Lastly, subjects will autonomously collect a fecal
        sample within 6 to 48h after the intervention.
  
        On the morning of the last day, after the last urine collection, subjects will bring
        collected urine and fecal samples and the 5-day food diary to the Human Nutrition Unit of the
        Food and Drug Department of the University of Parma.
  
        After the first arm, subjects will resume their habitual lifestyles for 2 consecutive weeks,
        subsequently, they will attend another visit, when all the anthropometric evaluations carried
        out during the first visit will be repeated and another kit with foods and information for
        the personalized diet will be given. With the study being a cross-over design, the protocol
        followed will be the same as the one described above (except for saliva sampling, which will
        only be performed once). Coming the third day of diet, subjects who ingested coffee in the
        prior arm of the study will ingest water, and vice-versa.
  
        For this study, the sample size was estimated considering the mean concentration of
        coffee-derived (poly)phenols and their metabolites in urine as the primary outcome and the
        available literature on similar approaches and studies of the area. The number of 30
        volunteers was established considering the accepted parameters for clinical trials (power set
        at 0.8 and α set at 0.05) possible dropouts (15% drop-out rate), and exclusion of volunteers
        to maintain the sample power for statistical analysis. Before any comparison is performed,
        the normality of each variable will be evaluated to choose the most appropriate statistical
        test. If metabolite data are normally distributed, data will be expressed as mean ± standard
        deviation and analysed using repeated measures t-test or ANOVA with post-hoc comparisons. If
        data are not normally distributed, they will be reported as median and Interquartile Range
        and the Friedman test with post hoc pairwise comparisons will be performed. P-value < 0.05
        will be regarded as statistically significant.
  
        Unsupervised and supervised multivariate analysis will be used to identify and characterize
        the different urinary metabotypes, following the procedures developed by our research group
        for targeted metabolomics analysis of phenolic metabolites or when using untargeted
        metabolomics data. We will be using different clustering approaches to better explore the
        existing variability. This will serve to establish the number of clusters (i.e., phenolic
        metabotypes), the number of participants forming each cluster and the similarities among
        possible metabotypes.
  
        The relationship between patterns of LMWP and their putative determinants (age, sex,
        ethnicity, lifestyle, anthropometrics, body composition, genotype, and gut microbiome) will
        be explored using correlation tests and multivariate analyses. P-value < 0.05 will be
        regarded as statistically significant. Statistics will be carried out by the specialized
        staff of the Human Nutrition Unit, under the supervision of the PI, and in collaboration with
        the University teams in charge of microbial profiling and genotyping.
  allocation: Randomized
  intervention model: Crossover Assignment
  primary purpose: Screening
  masking: Triple (Participant, Investigator, Outcomes Assessor)
  measure: Number and concentration of phenolic metabolites in urine
  time frame: Samples acquired from 60 hours prior to 48 hours after intervention or control
  description: Urine samples will be subjected to both targeted and untargeted liquid chromatography mass spectrometry analyses
  measure: Identification and quantification of gut bacteria (genera and species) in feces
  time frame: Samples acquired from 6-48 hours after coffee or control
  description: Analysis of microbiota taxonomy at species level from bacterial DNA extracted from fecal samples.
  measure: Number and concentration of phenolic metabolites in feces
  time frame: Samples acquired from 6-48 hours after coffee or control
  description: Fecal samples will be subjected to both targeted and untargeted liquid chromatography mass spectrometry analyses
  measure: Identification and quantification of metabolites after in vitro fecal fermentation
  time frame: Samples acquired from 6-48 hours after coffee or control
  description: A set of (poly)phenol-rich substrates and protein-rich food items will be incubated and fermented in sealed jars to evaluate the ability of human microbiota to produce different phenolic metabolites.
  measure: Identification of single-nucleotide polymorphism analysis
  time frame: Single saliva samples collected from 1 hour to 30 minutes before the intervention with coffee or control.
  description: Genotyping for possibile genetic variations in genes related to (poly)phenol kinetics. Commercially available SNP arrays detect gene variability across the majority of the genome using a tagging SNP approach.
  arm group label: Coffee
  arm group type: Experimental
  description: In this arm, volunteers will receive, once, 3 decaffeinated coffees (150 mL total) with sugar as a known source of (poly)phenols.
  arm group label: Control
  arm group type: Other
  description: In this arm, volunteers will receive, once, 150 mL of sugared water as control.
  intervention type: Other
  intervention name: Coffee
  description: Decaffeinated coffee with 1 pack (5g) of sugar from commercially available brands prepared with commercially available domestic coffee machines.
  arm group label: Coffee
  intervention type: Other
  intervention name: Water
  description: 150 mL of hot water with 1 pack (5g) of sugar from commercially available brands prepared with commercially available domestic coffee machines.
  arm group label: Control
  criteria: 
  gender: All
  minimum age: 20 Years
  maximum age: 40 Years
  healthy volunteers: Accepts Healthy Volunteers
  last name: Pedro M Mena Parreño, PhD
  role: Principal Investigator
  affiliation: University of Parma
  last name: Pedro M Mena Parreño, PhD
  phone: +393288087332
  email: pedromiguel.menaparreno@unipr.it
  last name: Letizia Bresciani, PhD
  email: letizia.bresciani@unipr.it
  facility: 
  country: Italy
  responsible party type: Principal Investigator
  investigator affiliation: University of Parma
  investigator full name: Pedro M. Mena Parreño
  investigator title: Principal Investigator
  sharing ipd: Yes
  ipd description: The data shared will be those that underlie results in a publication.
  ipd info type: Study Protocol
  ipd info type: Statistical Analysis Plan (SAP)
  ipd time frame: Data will be available from when published in peer-reviewed journals.
  ipd access criteria: Data available in publications can be freely accessed. If further data is required to be shared, it will be assessed case by case.