Handling and detection of 25 amol of near-infrared dye deoxynucleotide conjugates by capillary electrophoresis with laser-induced fluorescence detection

Free radicals and other reactive species can cause oxidative damage to biomolecules when oxidant species exceed the antioxidant defences in the body, resulting in oxidative stress. Oxidatively damaged products have been associated with aging as well as with the development of pathologies like cancer, cardiovascular disease, neurodegenerative disorders, diabetes, inflammation, etc. Reliable measurements of biomarkers of oxidative damage to macromolecules would afford information on the predisposition and prognosis of certain pathologies, being of utmost importance in evaluation of the effect of intervention with antioxidants on the incidence of diseases associated to oxidative stress. This review will present and compare different analytical methods, especially those involving chromatographic and electrophoretic techniques, commonly used for the analysis of biomarkers of oxidative damage to the three main macromolecules, namely oxidised DNA, lipid peroxidation products, and protein carbonyls.

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“…It is also highly sensitive, although it requires multiple reaction steps to form detectable nucleotide conjugates [45].…”

Section: Post-labelling Techniquesmentioning

confidence: 99%

Chromatographic and electrophoretic methods for the analysis of biomarkers of oxidative damage to macromolecules (DNA, lipids, and proteins)

Mateos

Bravo²

2007

J of Separation Science

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“…NP 1 is widely used for digesting DNA [16], however, the routinely used digestion mixture contains sodium acetate pH 5.5, which is subject to interferences since carboxylic acid groups also will be labeled. Therefore, MES buffer pH 5.5 was used.…”

Section: Comparative Ce-lif Analysis Of Ct-dna Digested To Pdns or Tomentioning

confidence: 99%

Detection and separation of nucleoside‐5'‐monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser‐induced fluorescence detection

et al. 2005

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We investigated the separation and detection of the 5'-monophosphates of 2'-deoxynucleosides selectively conjugated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) at the 5'-phosphate group using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). BODIPY conjugates of the four common deoxynucleoside-5'-monophosphates (2'-deoxyguanosine-5'-monophosphate, 2'-deoxyadenosine-5'-monophosphate, 2'-deoxycytidine-5'-monophosphate, and thymidine-5'-monophosphate) were prepared and subjected to CE-LIF to serve as standard compounds for peak assignment and to develop separation conditions for the analysis of DNA. BODIPY conjugates were detected and resolved by CE-LIF after digestion of DNA or an oligonucleotide to 5'-monophosphates by nuclease P1 (NP 1) and fluorescence labeling without further purification step. Comparative analyses of calf-thymus DNA digested either with micrococcal nuclease/spleen phosphodiesterase to 3'-monophosphates or with NP 1 to 5'-monophosphates showed that both versions of the fluorescence postlabeling assay were equally efficient and sensitive. Moreover, using the same assay, 2'-deoxyuridine and 2'-deoxy-5methylcytidine were identified in bisulfite treated DNA after NP 1 digestion indicating that fluorescence postlabeling of 2'-deoxyribonucleoside-5'-monophosphates with BODIPY FL EDA and detection by CE-LIF has the potential to determine DNA damage and genomic DNA methylation.

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“…More recently, capillary electrophoresis with laser-induced fluorescence (CE-LIF) has been employed for the study of DNA adducts [29,30]. Schmitz et al [31] have developed a CE-LIF method for studying a range of DNA adducts as carcinogenesis biomarkers, including 8-OHdG-3 -monophosphate (8-OH-dGMP).…”

Section: Fluorescent Labelingmentioning

confidence: 99%

Recent developments in analytical methodology for 8-hydroxy-2′-deoxyguanosine and related compounds

Peoples

Karnes

2005

Journal of Chromatography B

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Handling and detection of 25 amol of near-infrared dye deoxynucleotide conjugates by capillary electrophoresis with laser-induced fluorescence detection

Cited by 11 publications

References 10 publications

Chromatographic and electrophoretic methods for the analysis of biomarkers of oxidative damage to macromolecules (DNA, lipids, and proteins)

Chromatographic and electrophoretic methods for the analysis of biomarkers of oxidative damage to macromolecules (DNA, lipids, and proteins)

Detection and separation of nucleoside‐5'‐monophosphates of DNA by conjugation with the fluorescent dye BODIPY and capillary electrophoresis with laser‐induced fluorescence detection

Recent developments in analytical methodology for 8-hydroxy-2′-deoxyguanosine and related compounds

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