<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/NCT02212288</url>
  </required_header>
  <id_info>
    <org_study_id>PHAO14/FM-StressOX-PCU</org_study_id>
    <nct_id>NCT02212288</nct_id>
  </id_info>
  <brief_title>Antioxidant Signature in Adult Patients With Phenylketonuria</brief_title>
  <acronym>StressOX-PCU</acronym>
  <official_title>Antioxidant Signature in Adult Patients With Phenylketonuria (PKU) and Healthy Volunteers : Relationship With Metabolic Disturbances</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>University Hospital, Tours</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
    <collaborator>
      <agency>Institut National de la Santé Et de la Recherche Médicale, France</agency>
      <agency_class>Other</agency_class>
    </collaborator>
  </sponsors>
  <source>University Hospital, Tours</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
  </oversight_info>
  <brief_summary>
    <textblock>
      To date, oxidative stress in PKU has been evaluated only with fragmented approaches.The aim&#xD;
      of the present study is to investigate oxidative stress in PKU with more comprehensive&#xD;
      methods.The relationship between oxidative stress and metabolic disturbances&#xD;
      (hyperPhenylalaninemia) will also be studied.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Analysis of oxidative stress&#xD;
&#xD;
      Oxidative metabolism is most likely disrupted leukocytes in the PCU thereof having DNA breaks&#xD;
      correlated to blood Phe levels (22). Accessibility by patients, these cells are therefore&#xD;
      interesting &quot;tools&quot; for analysis of oxidative stress in PKU.&#xD;
&#xD;
      -Hydrogen Peroxide intraleucocytaire We will isolate the leukocytes from heparinized samples&#xD;
      to analyze intracellular H2O2 levels by flow cytometry after labeling with 5-(and&#xD;
      6)-chloromethyl-2 ', 7'-dicholorohydrofluorescein diacetate, acetyl ester (CM-H2DCFDA).&#xD;
&#xD;
      Anti-oxydogramme We will carry out an anti-oxydogramme which was the subject of a patent&#xD;
      (Herault O &amp; Vignon C &quot;Method for Cancer Diagnosis&quot; - Patent PCT / EP2011 / 073,757 filed&#xD;
      Dec. 22, 2011) in the field of oncology. This is to achieve expression of an anti-oxidant&#xD;
      genes in PKU profile compared with control subjects. This approach is based on three&#xD;
      techniques: determining the level of expression of key antioxidants by RT-qPCR gene&#xD;
      comparison with the expression of a housekeeping gene (GAPDH), ranked by their level of gene&#xD;
      expression and comparison with the level of expression of the same genes in a reference pool&#xD;
      RNA, used as internal calibrator. We will express these results through the RQ (relative&#xD;
      quantification). The panel of antioxidant genes studied is the following: genes antioxidant&#xD;
      enzyme systems not thiols: superoxide dismutase (SOD1, SOD2, SOD3) and catalase (CAT) or&#xD;
      thiols: glutathione system (GPX, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 GSR), the&#xD;
      thioredoxin (TXN, TXN2) of peroxyredoxins (PRDX, PRDX2, PRDX4, PRDX5, PRDX6) and&#xD;
      glutaredoxins (GLRX, GLRX2, GLRX3, GLRX5).&#xD;
&#xD;
        -  Way NRF2 The NRF2 pathway H2O2 / being very classically activated when oxidative stress,&#xD;
           we will quantify the expression of target genes NRF2 (HMOX, NQO1, GPX2). If they are&#xD;
           overexpressed, the nuclear translocation of NRF2 be studied by confocal microscopy.&#xD;
&#xD;
        -  DNA Breaks The breaks in DNA related to the oxidative stress in leukocytes are analyzed&#xD;
           by flow cytometry (marking -H2AX) and electrophoretic migration of the fluorescent DNA&#xD;
           (COMET assay).&#xD;
&#xD;
        -  antioxidant vitamins and trace elements We will measure the plasma concentrations of&#xD;
           vitamin A, C and E by a technique of liquid chromatography with UV detection.&#xD;
           Furthermore, the activity of many enzymes is dependent antioxidant micronutrient, we&#xD;
           doserons plasma concentrations of Se, Cu, Mn, and Zn.&#xD;
&#xD;
      Metabolic Balance&#xD;
&#xD;
      We realize chromatography plasma amino acids using the standard method of ion exchange&#xD;
      chromatography with post-column derivation with ninhydrin. We will evaluate in particular the&#xD;
      concentration of phenylalanine and tyrosine. We will focus on also other amino acids as&#xD;
      described antioxidant potential (citrulline, taurine, histidine ...) We will evaluate by&#xD;
      GC-MS organic acids commonly accumulated in the urine of PKU patients:&#xD;
      dihydroxyphenylalanine, phenyllactate, phenylpyruvate, phenylacetate. Bioptérines&#xD;
      concentrations and its derivatives will also be evaluated in HPLC with fluorescence&#xD;
      detection.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date>September 2014</start_date>
  <completion_date type="Actual">January 2015</completion_date>
  <primary_completion_date type="Actual">January 2015</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Case-Control</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>Plasma concentrations of oxidative stress markers</measure>
    <time_frame>First Day</time_frame>
    <description>The main purpose of this preliminary study is to characterize oxidative stress in patients with PKU adults through a comprehensive approach involving the assessment of a wide combination of characteristic markers of oxidative stress and metabolic parameters that may be involved. In this context, we propose to establish an anti-oxidant signing in adult patients PCU using an innovative and comprehensive technical involving&#xD;
Quantification of intraleucocytaire hydrogen peroxide (Evaluated by flow cytometry and is expressed as relative fluorescence intensity between patients and the control group)&#xD;
Study of the expression of 24 genes involved in oxidative stress response (anti-oxydogramme)&#xD;
Comparison nuclear NRF2 translocation&#xD;
Comparison of DNA breaks&#xD;
Quantification of antioxidant vitamins and trace elements.(in difference from Normal values)</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Plasma concentrations of amino acids and organic acids concentration</measure>
    <time_frame>First day</time_frame>
    <description>Measurement of concentration of amino acids and organic acids concentration found in the 2 groups in comparison to normal values</description>
  </secondary_outcome>
  <number_of_groups>2</number_of_groups>
  <enrollment type="Actual">20</enrollment>
  <condition>PKU</condition>
  <condition>Hyperphenylalaninemia</condition>
  <arm_group>
    <arm_group_label>PKU patients</arm_group_label>
    <description>Adult PKU patients;Blood samples;Urine sample only at inclusion</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Healthy controls</arm_group_label>
    <description>Adult Healthy controls;Blood samples;Urine samples only at inclusion</description>
  </arm_group>
  <intervention>
    <intervention_type>Other</intervention_type>
    <intervention_name>Blood sample</intervention_name>
    <description>Blood samples are for metabolic and genetic and biological investigation</description>
    <arm_group_label>Healthy controls</arm_group_label>
    <arm_group_label>PKU patients</arm_group_label>
  </intervention>
  <intervention>
    <intervention_type>Other</intervention_type>
    <intervention_name>Urine sample</intervention_name>
    <description>Urine sample for biological investigation</description>
    <arm_group_label>Healthy controls</arm_group_label>
    <arm_group_label>PKU patients</arm_group_label>
  </intervention>
  <biospec_retention>Samples With DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      Blood samples&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        10 PKU patients and 10 healthy controls with age and sex match&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  PKU patients over 18 years of age with phenylketonuria not treated with a&#xD;
             phenylalanine-controlled diet.&#xD;
&#xD;
          -  Affiliation to a social security scheme&#xD;
&#xD;
          -  Reading and signing the informed consent&#xD;
&#xD;
          -  Control subjects: all healthy subjects over 18 years of unfulfilled abnormal&#xD;
             metabolism of whatever nature and associated symptoms&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  Pregnancy of patients and control subjects women (confirmed by a negative pregnancy&#xD;
             test)&#xD;
&#xD;
          -  Patients with diseases that may cause oxidative stress (neurodegenerative diseases,&#xD;
             cancer)&#xD;
&#xD;
          -  Patients treated with drugs known to cause oxidative stress&#xD;
&#xD;
          -  Control subjects:controlled protein diet&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>65 Years</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>François MAILLOT, MD-PhD</last_name>
    <role>Study Director</role>
    <affiliation>University Hospital of TOURS, France</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>University Hospital of TOURS</name>
      <address>
        <city>Tours</city>
        <state>I&amp;L</state>
        <zip>37044</zip>
        <country>France</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>France</country>
  </location_countries>
  <verification_date>January 2017</verification_date>
  <study_first_submitted>July 28, 2014</study_first_submitted>
  <study_first_submitted_qc>August 6, 2014</study_first_submitted_qc>
  <study_first_posted type="Estimate">August 8, 2014</study_first_posted>
  <last_update_submitted>January 13, 2017</last_update_submitted>
  <last_update_submitted_qc>January 13, 2017</last_update_submitted_qc>
  <last_update_posted type="Estimate">January 16, 2017</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor</responsible_party_type>
  </responsible_party>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Phenylketonurias</mesh_term>
  </condition_browse>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


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/NCT02212288
org study id: PHAO14/FM-StressOX-PCU
nct id: NCT02212288
lead sponsor: 
collaborator: 
has dmc: No
To date, oxidative stress in PKU has been evaluated only with fragmented approaches.The aim
      of the present study is to investigate oxidative stress in PKU with more comprehensive
      methods.The relationship between oxidative stress and metabolic disturbances
      (hyperPhenylalaninemia) will also be studied.
Analysis of oxidative stress

      Oxidative metabolism is most likely disrupted leukocytes in the PCU thereof having DNA breaks
      correlated to blood Phe levels (22). Accessibility by patients, these cells are therefore
      interesting "tools" for analysis of oxidative stress in PKU.

      -Hydrogen Peroxide intraleucocytaire We will isolate the leukocytes from heparinized samples
      to analyze intracellular H2O2 levels by flow cytometry after labeling with 5-(and
      6)-chloromethyl-2 ', 7'-dicholorohydrofluorescein diacetate, acetyl ester (CM-H2DCFDA).

      Anti-oxydogramme We will carry out an anti-oxydogramme which was the subject of a patent
      (Herault O & Vignon C "Method for Cancer Diagnosis" - Patent PCT / EP2011 / 073,757 filed
      Dec. 22, 2011) in the field of oncology. This is to achieve expression of an anti-oxidant
      genes in PKU profile compared with control subjects. This approach is based on three
      techniques: determining the level of expression of key antioxidants by RT-qPCR gene
      comparison with the expression of a housekeeping gene (GAPDH), ranked by their level of gene
      expression and comparison with the level of expression of the same genes in a reference pool
      RNA, used as internal calibrator. We will express these results through the RQ (relative
      quantification). The panel of antioxidant genes studied is the following: genes antioxidant
      enzyme systems not thiols: superoxide dismutase (SOD1, SOD2, SOD3) and catalase (CAT) or
      thiols: glutathione system (GPX, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 GSR), the
      thioredoxin (TXN, TXN2) of peroxyredoxins (PRDX, PRDX2, PRDX4, PRDX5, PRDX6) and
      glutaredoxins (GLRX, GLRX2, GLRX3, GLRX5).

        -  Way NRF2 The NRF2 pathway H2O2 / being very classically activated when oxidative stress,
           we will quantify the expression of target genes NRF2 (HMOX, NQO1, GPX2). If they are
           overexpressed, the nuclear translocation of NRF2 be studied by confocal microscopy.

        -  DNA Breaks The breaks in DNA related to the oxidative stress in leukocytes are analyzed
           by flow cytometry (marking -H2AX) and electrophoretic migration of the fluorescent DNA
           (COMET assay).

        -  antioxidant vitamins and trace elements We will measure the plasma concentrations of
           vitamin A, C and E by a technique of liquid chromatography with UV detection.
           Furthermore, the activity of many enzymes is dependent antioxidant micronutrient, we
           doserons plasma concentrations of Se, Cu, Mn, and Zn.

      Metabolic Balance

      We realize chromatography plasma amino acids using the standard method of ion exchange
      chromatography with post-column derivation with ninhydrin. We will evaluate in particular the
      concentration of phenylalanine and tyrosine. We will focus on also other amino acids as
      described antioxidant potential (citrulline, taurine, histidine ...) We will evaluate by
      GC-MS organic acids commonly accumulated in the urine of PKU patients:
      dihydroxyphenylalanine, phenyllactate, phenylpyruvate, phenylacetate. Bioptérines
      concentrations and its derivatives will also be evaluated in HPLC with fluorescence
      detection.
observational model: Case-Control
time perspective: Prospective
measure: Plasma concentrations of oxidative stress markers
time frame: First Day
description: The main purpose of this preliminary study is to characterize oxidative stress in patients with PKU adults through a comprehensive approach involving the assessment of a wide combination of characteristic markers of oxidative stress and metabolic parameters that may be involved. In this context, we propose to establish an anti-oxidant signing in adult patients PCU using an innovative and comprehensive technical involving
Quantification of intraleucocytaire hydrogen peroxide (Evaluated by flow cytometry and is expressed as relative fluorescence intensity between patients and the control group)
Study of the expression of 24 genes involved in oxidative stress response (anti-oxydogramme)
Comparison nuclear NRF2 translocation
Comparison of DNA breaks
Quantification of antioxidant vitamins and trace elements.(in difference from Normal values)
measure: Plasma concentrations of amino acids and organic acids concentration
time frame: First day
description: Measurement of concentration of amino acids and organic acids concentration found in the 2 groups in comparison to normal values
arm group label: PKU patients
description: Adult PKU patients;Blood samples;Urine sample only at inclusion
arm group label: Healthy controls
description: Adult Healthy controls;Blood samples;Urine samples only at inclusion
intervention type: Other
intervention name: Blood sample
description: Blood samples are for metabolic and genetic and biological investigation
arm group label: Healthy controls
arm group label: PKU patients
intervention type: Other
intervention name: Urine sample
description: Urine sample for biological investigation
arm group label: Healthy controls
arm group label: PKU patients
Blood samples
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: All
minimum age: 18 Years
maximum age: 65 Years
healthy volunteers: Accepts Healthy Volunteers
last name: François MAILLOT, MD-PhD
role: Study Director
affiliation: University Hospital of TOURS, France
facility: 
country: France
responsible party type: Sponsor
mesh term: Phenylketonurias