NCT02212288

Antioxidant Signature in Adult Patients With Phenylketonuria

Antioxidant Signature in Adult Patients With Phenylketonuria (PKU) and Healthy Volunteers : Relationship With Metabolic Disturbances

• Status: Completed
• Phase: —
• Study type: Observational
• Enrollment: 20 (actual)
• Sponsor: University Hospital, Tours · Academic / Other
• Sex: All
• Age: 18 Years – 65 Years
• Healthy volunteers: Accepted

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.

Conditions

• PKU
• Hyperphenylalaninemia

Interventions

Type	Name	Description
OTHER	Blood sample	Blood samples are for metabolic and genetic and biological investigation
OTHER	Urine sample	Urine sample for biological investigation

Timeline

• Start date: 2014-09-01
• Primary completion: 2015-01-01
• Completion: 2015-01-01
• First posted: 2014-08-08
• Last updated: 2017-01-16

Locations

1 site across 1 country: France