Capillary electrophoresis-laser induced fluorescence analysis of endogenous damage in mitochondrial and genomic DNA

Reactive oxygen molecules are formed in vivo as by‐products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have develope...

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Bibliographic Details
Published in:Electrophoresis 2005-06, Vol.26 (13), p.2599-2607
Main Authors: Wirtz, Michaela, Schumann, Claus A., Schellenträger, Marc, Gäb, Siegmar, vom Brocke, Jochen, Podeschwa, Michael A. L., Altenbach, Hans-J., Oscier, David, Schmitz, Oliver J.
Format: Article
Language:English
Subjects:
5-Methylcytosine - analysis
5-Methylcytosine - isolation & purification
Adenosine Monophosphate - analogs & derivatives
Adenosine Monophosphate - analysis
Adenosine Monophosphate - isolation & purification
Animals
Biomarkers - analysis
Capillary electrophoresis
Cattle
Chromatography, Micellar Electrokinetic Capillary - methods
DNA adducts
DNA Adducts - analysis
DNA Adducts - isolation & purification
DNA Damage
DNA Methylation
DNA, Mitochondrial - chemistry
Electrophoresis, Capillary - methods
Genome
Humans
Laser-induced fluorescence
Lasers
Leukemia, Lymphocytic, Chronic, B-Cell - metabolism
Liver - chemistry
Oxidative Stress
Spectrometry, Fluorescence
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Summary:Reactive oxygen molecules are formed in vivo as by‐products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have developed an analytical method that involves the isolation and hydrolysis of genomic DNA or mtDNA, the labeling of modified and unmodified nucleotides and micellar electrokinetic chromatography with laser‐induced fluorescence detection. With this method we have found etheno‐adenine, thymine glycol, uracil, hypoxanthine, and 5‐methylcytosine. These were identified by the addition of internal standards to the genomic or mtDNA. There are a large number of other signals in the electropherograms of mtDNA that we have never found in genomic DNA analysis because they are at lower concentration in the genome. In the DNA of untreated patients with chronic lymphocytic leukemia (CLL), uracil and high levels of etheno‐adenine were found, which can be explained by antioxidant enzyme alterations and oxidative stress in the CLL lymphocytes.
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.200410397