Antioxidant Signature in Adult Patients With Phenylketonuria

NCT ID: NCT02212288

Last Updated: 2017-01-16

Basic Information

• Recruitment Status: COMPLETED
• Total Enrollment: 20 participants
• Study Classification: OBSERVATIONAL
• Study Start Date: 2014-09-30
• Study Completion Date: 2015-01-31

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.

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

Study Design

• Observational Model Type: CASE_CONTROL
• Study Time Perspective: PROSPECTIVE

Study Groups

PKU patients

Adult PKU patients;Blood samples;Urine sample only at inclusion

Blood sample

Intervention Type: OTHER

Blood samples are for metabolic and genetic and biological investigation

Urine sample

Intervention Type: OTHER

Urine sample for biological investigation

Healthy controls

Adult Healthy controls;Blood samples;Urine samples only at inclusion

Blood sample

Intervention Type: OTHER

Blood samples are for metabolic and genetic and biological investigation

Urine sample

Intervention Type: OTHER

Urine sample for biological investigation

Interventions

Blood sample

Blood samples are for metabolic and genetic and biological investigation

Intervention Type: OTHER

Urine sample

Urine sample for biological investigation

Intervention Type: OTHER

Eligibility Criteria

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

• Minimum Eligible Age: 18 Years
• Maximum Eligible Age: 65 Years
• Eligible Sex: ALL
• Accepts Healthy Volunteers: Yes

Sponsors

Institut National de la Santé Et de la Recherche Médicale, France

OTHER_GOV

Sponsor Role: collaborator

University Hospital, Tours

OTHER

Sponsor Role: lead

Responsible Party

Responsibility Role: SPONSOR

Principal Investigators

François MAILLOT, MD-PhD

Role: STUDY_DIRECTOR

University Hospital of TOURS, France

Locations

University Hospital of TOURS

Tours, I&L, France

Countries

France

Other Identifiers

PHAO14/FM-StressOX-PCU

Identifier Type: -

Identifier Source: org_study_id