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.

Keywords: 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxo-7,8-dihydroguanosine; LC-MS/MS; pre-heat.

FIGURE 1

The effect of pre‐heat after storing at −80°C for 1 week in healthy individuals 8‐oxoG, 8‐oxo‐7,8‐dihydroguanosine; 8‐oxodG, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine

FIGURE 2

The effect of pre‐heat after storing at −80°C for 1 month in 18 individuals 8‐oxoG, 8‐oxo‐7,8‐dihydroguanosine; 8‐oxodG, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine