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

Abstract

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.

RNA analysis by MEKC with LIF detection

“…The values show a similar sensitivity compared with the related analysis of DNA bases using the same method [5,6] with a LOD of the method for the analysis of RNA based on BODIPY conjugates of nucleoside-5'-monophosphates in the range from 80 to 200 pM determined by dilution of derivatized 10 mg samples of digested oligonucleotides. Likewise, correction factors were similar to those obtained by Wirtz et al [7,10] for 2'-deoxyribonucleoside-3'-monophosphates except for I. Because correction factors depend on the size of the nucleic acid used for calibration [7,10] the correction factors shown represent a good approximation only.…”

“…Likewise, correction factors were similar to those obtained by Wirtz et al [7,10] for 2'-deoxyribonucleoside-3'-monophosphates except for I. Because correction factors depend on the size of the nucleic acid used for calibration [7,10] the correction factors shown represent a good approximation only. Highest correction factors were observed for G, I and X which are most likely due to an exceptionally weak fluorescence ac- tivity resulting from the electron transfer between the purine ring and the difluoro-bora-diazaindacen skeleton.…”

“…The migration times of analytes were normalized by division with the migration time of the adenosine-5'-monophosphate (pA)-BODIPY peak and signals were time-corrected as reported before [10].…”

“…For quantitative analyses individual correction factors of pNs that take into account differential yields of hydrolysis and labeling reactions as well as the differences in fluorescence quantum yields were determined as described before [7,9,10], except that site specifically modified oligoribonucleotides were used instead of Lambda-DNA.…”

“…In this way we have prepared and characterized BODIPY FL EDA conjugates of the four normal deoxyribonucleosides as 3'-and 5'-monophosphates [5,6] and used them as reference compounds for the analysis of DNA by CE-LIF. Additionally, 2'-deoxy-5-methylcytidine-3'-monophosphate was synthesized, derivatized with BODIPY FL EDA [7] and used as standard for the determination of the methylation level in human tumor cells [8,9] and mitochondrial DNA [10].…”