Antioxidant Signature in Adult Patients With Phenylketonuria
关键词
抽象
描述
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.
日期
最后验证: | 12/31/2016 |
首次提交: | 07/27/2014 |
提交的预估入学人数: | 08/05/2014 |
首次发布: | 08/07/2014 |
上次提交的更新: | 01/12/2017 |
最近更新发布: | 01/15/2017 |
实际学习开始日期: | 08/31/2014 |
预计主要完成日期: | 12/31/2014 |
预计完成日期: | 12/31/2014 |
状况或疾病
干预/治疗
Other: Blood sample
Other: Urine sample
相
手臂组
臂 | 干预/治疗 |
---|---|
PKU patients Adult PKU patients;Blood samples;Urine sample only at inclusion | |
Healthy controls Adult Healthy controls;Blood samples;Urine samples only at inclusion |
资格标准
有资格学习的年龄 | 18 Years 至 18 Years |
有资格学习的性别 | All |
取样方式 | Non-Probability Sample |
接受健康志愿者 | 是 |
标准 | 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 |
结果
主要结果指标
1. Plasma concentrations of oxidative stress markers [First Day]
次要成果指标
1. Plasma concentrations of amino acids and organic acids concentration [First day]