Antioxidant Signature in Adult Patients With Phenylketonuria
Condition: PKU; Hyperphenylalaninemia
MeSH terms: Phenylketonurias
Study type: Observational

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.
    

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.
    

Intervention name(s): Blood sample; Urine sample
Intervention type(s): Other; Other
Intervention description(s): Blood samples are for metabolic and genetic and biological investigation; Urine sample for biological investigation
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        10 PKU patients and 10 healthy controls with age and sex match
      
Biospecimen description: 
      Blood samples