Dataset Information

Is pre-heat necessary for the measurement of 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine samples.

ABSTRACT:

Background

It is currently unclear for the necessary of pre-heating urine samples for the accurate determination of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Thus, we conducted this study to evaluate the effect of pre-heat (i.e., to 37°C) on the accurate measurement of 8-oxoG and 8-oxodG in frozen urine samples.

Methods

Random urine samples from six healthy volunteers, six patients with renal dysfunction, and six patients with systematic diseases such as diabetes were collected, split, and stored at -80°C for up to 1 month. The frozen samples were thawed at room temperature (RT) or 37°C for different time, 10-fold diluted with ddH2O containing 1% formic acid, and determined by self-established LC-MS/MS method coupled with an ACQUITY™ Primer HSS T3 column.

Results

Thawing the samples at RT for 30 or 120 min, or at 37°C for 15 or 90 min did not affect the determination of 8-oxoG and 8-oxodG in urine samples. Moreover, no significant difference between thawing the urine samples at RT and 37°C was found after storing at -80°C for 1-3 months.

Conclusion

It is not always necessary to pre-heat the frozen urine samples to release 8-oxoG and 8-oxodG from precipitates, which is associated with different pre-treatment and determination methods.

SUBMITTER: Zou Y

PROVIDER: S-EPMC9550956 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Publications

Is pre-heat necessary for the measurement of 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine samples.

Zou Yutong Y Ma Xiaoli X Yu Songlin S Qiu Ling L

Journal of clinical laboratory analysis 20220829 10

<h4>Background</h4>It is currently unclear for the necessary of pre-heating urine samples for the accurate determination of 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Thus, we conducted this study to evaluate the effect of pre-heat (i.e., to 37°C) on the accurate measurement of 8-oxoG and 8-oxodG in frozen urine samples.<h4>Methods</h4>Random urine samples from six healthy volunteers, six patients with renal dysfunction, and six patients with systemati ...[more]

PMID: 36036744

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Project description:Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Dataset Information

Is pre-heat necessary for the measurement of 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine samples.

Background

Methods

Results

Conclusion

Publications

Is pre-heat necessary for the measurement of 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine samples.

Similar Datasets

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