Determination of 8-Hydroxy-2 Deoxyguanosine in Pseudomonas Fluorescens Freeze-Dried Exposed to the ROS Using HPLC with Electrochemical Detection Method
Abstract
Oxidative DNA dam age is involved in the f cell death induced by freeze -dried powder during storage. Cell 8 - hydroxy -2’deoxyguanosine (8 -oxodG) is widely accepted as a biomarker of the “freeze -dried bacteria” oxidative DNA damage. The aim of this study was to introduce a met hod for determination 8 -oxodG in cell freeze -dried samples using high - performance liquid chromatography with electrochemical detection. In the tested range of 0.5 µmol L-1 to 1.0 nmol L-1, the calibration curve was linear (r2=0.9995) and the limit of detec tion was 0.05 µmol L-1. The used method did not allow highlighting the presence in the samples of the 8OH within the limits of detection. A more successful method (more sensitive) would be needed to detect possibly the 8OH.
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Introduction
The drying by lyophilisation of microorganisms has some consequence on the viability of the latter. The powder of lyophilized bacteria remains vulnerable on the environmental conditions such as tempera ture, ultraviolet, reactive species of oxygens. The latter have mainly four consequences: the loss of the cellular viability caused by an oxidation of the biological materials like lipids, proteins and DNA [1,2,3] . The drying method cause severe damage in the cellular membranes such as the lipid peroxidation, denaturation of proteins and DNA leading to a loss of viability [4].
Freeze -drying and the storage of cells lead damage in proteins, fatty acids and DNA [5, 6] . Although the DNA damage undergone by the cellular membrane during the freeze -drying. It still plays an essential role in the loss of viability. The damage of the cellular components DNA and ARN affects considerably the viability of the freeze -dried bacteria during storage. The DNA is very sensitive to the drying, as demonstrated on Echericha coli [7, 8] .
There are numerous modifications observed after the oxidation of the DNA, the major product of DNA oxidative damage is 8-hydroxyguanine (8 -oxoG) and its nucleoside 8 -hydroxy -2’deoxyguanosine (8 -oxodG). 8 -oxodG has widely been accepted as a biomarker of DNA damage and cellular oxidative stress [9].
The present study will be focus on the development of novel method for the detection and quantification of the 2 - déoxyguanosine 2dG and the 8 -hydroxy -2-déoxyguanosine 8OH in extracts of DNA bacterial of Pseudomonas fluorescens freeze -dried. Because the 8 -hydroxy -2-déoxyguanosine is the main oxidized by -product of the DNA used as a biomarker of the oxidative stress.
Conclusion
The main purpose of this study was to confirm the hypothesis which supported that DNA also is subject of the oxidation in the same manner as lipids and proteins. The sensitivity of the 2dG was better measured in our operating conditions compared to the 8OH one. T he current method was not able to highlight within the limits of detection the presence of the 8OH sa mples. An additional method would be needed for such to detection. Some scientists describe the tests on the oxidation of the ARN instead of the DNA, which represent an interesting way to explore [8].
