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

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

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.

Study Overview

• Status: Completed
• Conditions: PKU; Hyperphenylalaninemia
• Intervention / Treatment: 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.

• Study Type: Observational
• Enrollment (Actual): 20

Contacts and Locations

Study Locations

• France
  • I&L
    • Tours, I&L, France, 37044
      • University Hospital of Tours

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 65 years (Adult, Older Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

• Sampling Method: Non-Probability Sample

Study Population

10 PKU patients and 10 healthy controls with age and sex match

Description

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

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

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

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Plasma concentrations of oxidative stress markers	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)	First Day

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Plasma concentrations of amino acids and organic acids concentration	Measurement of concentration of amino acids and organic acids concentration found in the 2 groups in comparison to normal values	First day

Collaborators and Investigators

• Sponsor: University Hospital, Tours
• Collaborators: Institut National de la Santé Et de la Recherche Médicale, France
• Investigators: Study Director: François MAILLOT, MD-PhD, University Hospital of TOURS, France

Study record dates

Study Major Dates

• Study Start: September 1, 2014
• Primary Completion (Actual): January 1, 2015
• Study Completion (Actual): January 1, 2015

Study Registration Dates

• First Submitted: July 28, 2014
• First Submitted That Met QC Criteria: August 6, 2014
• First Posted (Estimate): August 8, 2014

Study Record Updates

• Last Update Posted (Estimate): January 16, 2017
• Last Update Submitted That Met QC Criteria: January 13, 2017
• Last Verified: January 1, 2017

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Metabolic Diseases; Brain Diseases; Central Nervous System Diseases; Nervous System Diseases; Genetic Diseases, Inborn; Metabolism, Inborn Errors; Brain Diseases, Metabolic; Brain Diseases, Metabolic, Inborn; Amino Acid Metabolism, Inborn Errors; Phenylketonurias
• Other Study ID Numbers: PHAO14/FM-StressOX-PCU