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Antioxidant Signature in Adult Patients With Phenylketonuria (StressOX-PCU)

The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details. Identifier: NCT02212288
Recruitment Status : Completed
First Posted : August 8, 2014
Last Update Posted : January 16, 2017
Institut National de la Santé Et de la Recherche Médicale, France
Information provided by (Responsible Party):
University Hospital, Tours

Brief Summary:
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.

Condition or disease Intervention/treatment
PKU Hyperphenylalaninemia Other: Blood sample Other: Urine sample

Detailed Description:

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.

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Study Type : Observational
Actual Enrollment : 20 participants
Observational Model: Case-Control
Time Perspective: Prospective
Official Title: Antioxidant Signature in Adult Patients With Phenylketonuria (PKU) and Healthy Volunteers : Relationship With Metabolic Disturbances
Study Start Date : September 2014
Actual Primary Completion Date : January 2015
Actual Study Completion Date : January 2015

Group/Cohort Intervention/treatment
PKU patients
Adult PKU patients;Blood samples;Urine sample only at inclusion
Other: Blood sample
Blood samples are for metabolic and genetic and biological investigation

Other: Urine sample
Urine sample for biological investigation

Healthy controls
Adult Healthy controls;Blood samples;Urine samples only at inclusion
Other: Blood sample
Blood samples are for metabolic and genetic and biological investigation

Other: Urine sample
Urine sample for biological investigation

Primary Outcome Measures :
  1. Plasma concentrations of oxidative stress markers [ Time Frame: First Day ]

    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

    1. Quantification of intraleucocytaire hydrogen peroxide (Evaluated by flow cytometry and is expressed as relative fluorescence intensity between patients and the control group)
    2. Study of the expression of 24 genes involved in oxidative stress response (anti-oxydogramme)
    3. Comparison nuclear NRF2 translocation
    4. Comparison of DNA breaks
    5. Quantification of antioxidant vitamins and trace elements.(in difference from Normal values)

Secondary Outcome Measures :
  1. Plasma concentrations of amino acids and organic acids concentration [ Time Frame: First day ]
    Measurement of concentration of amino acids and organic acids concentration found in the 2 groups in comparison to normal values

Biospecimen Retention:   Samples With DNA
Blood samples

Information from the National Library of Medicine

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Ages Eligible for Study:   18 Years to 65 Years   (Adult, Older Adult)
Sexes Eligible for Study:   All
Accepts Healthy Volunteers:   Yes
Sampling Method:   Non-Probability Sample
Study Population
10 PKU patients and 10 healthy controls with age and sex match

Inclusion Criteria:

  • PKU patients over 18 years of age with phenylketonuria not treated with a phenylalanine-controlled diet.
  • Affiliation to a social security scheme
  • Reading and signing the informed consent
  • Control subjects: all healthy subjects over 18 years of unfulfilled abnormal metabolism of whatever nature and associated symptoms

Exclusion Criteria:

  • Pregnancy of patients and control subjects women (confirmed by a negative pregnancy test)
  • Patients with diseases that may cause oxidative stress (neurodegenerative diseases, cancer)
  • Patients treated with drugs known to cause oxidative stress
  • Control subjects:controlled protein diet

Information from the National Library of Medicine

To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.

Please refer to this study by its identifier (NCT number): NCT02212288

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University Hospital of TOURS
Tours, I&L, France, 37044
Sponsors and Collaborators
University Hospital, Tours
Institut National de la Santé Et de la Recherche Médicale, France
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Study Director: François MAILLOT, MD-PhD University Hospital of TOURS, France
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Responsible Party: University Hospital, Tours Identifier: NCT02212288    
Other Study ID Numbers: PHAO14/FM-StressOX-PCU
First Posted: August 8, 2014    Key Record Dates
Last Update Posted: January 16, 2017
Last Verified: January 2017
Additional relevant MeSH terms:
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Brain Diseases, Metabolic, Inborn
Brain Diseases, Metabolic
Brain Diseases
Central Nervous System Diseases
Nervous System Diseases
Amino Acid Metabolism, Inborn Errors
Metabolism, Inborn Errors
Genetic Diseases, Inborn
Metabolic Diseases