Reply to commentary on "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

We thoroughly read the commentary from Hu et al. to our recently published article1 and admire their work. To make our point clearer and better clarify the necessity of pre-heat for the accurate measurement of 8- oxo- 7,8-dihydroguanosine (8-oxoG) and 8- oxo- 7,8- dihydro- 2′ - deoxyguanosine (8-oxodG) in urine samples, we give some more detailed instructions, which are shown as follows. (1) The conclusion “it is not always necessary to pre-heat the frozen urine samples to release 8-oxoG and 8-oxodG from precipitates” is only applicable for some certain situations, such as thorough mix and dilution (e.g., at least five times).2 As we emphasized in the article,1 this necessary is closely associated with different pretreatment and determination methods. Our results showed that 11-times dilution can completely release 8-oxoG and 8-oxodG from precipitations. Thus, whether the process of heating urine samples for the release of 8-oxoG and 8-oxodG is necessary and the specific details should be evaluated under different pretreatment and determination methods. (2) Thorough mix of the precipitations in urine samples after thawing is very important, which could guarantee that each aliquot (10 μL in this study) has a homogeneous distribution of precipitations in suspension. We would thoroughly successively mix all urine samples after thawing without discarding precipitations, and specially vortex it for several seconds before we draw every sample for further dilution. We have established and applied the LC–MS/MS method for the determination of 8-oxoG and 8-oxodG in urine samples for several years,3 and more than two researchers in our laboratory have validated the unnecessary to pre-heat the frozen urine samples to release 8-oxoG and 8-oxodG from precipitations with this pretreatment methods. Moreover, the verification results including precision were deemed good with the total coefficient of variation <4.2%.4 (3) To obtain more accurate measurements, the fresh urine samples without precipitations are first recommended. In this study1 and previous reports,3, 4 we all collected fresh urine samples, which are drawn at the same day; thus, there were no apparent precipitations in the urine samples before aliquoted and frozen. After storing at −20°C or − 80°C, most of them would form precipitations. Since diluting the urine 11-times with ddH2O containing 1% formic acid could completely release 8-oxoG and 8-oxodG from urinary precipitations, it is very important to thoroughly mix the samples and draw the needed volume with homogeneous distribution of precipitations as soon as possible. If the operators could not guarantee their performance and heater instruments are available, they should pre-heat the frozen urine samples to completely release the 8-oxoG and 8-oxodG from precipitations. This work was funded by research grants from the National High Level Hospital Clinical Research Funding (2022-PUMCH-B-073) and National High Level Hospital Clinical Research Funding (2022-PUMCH-A-138). The authors declare that they have no competing interests.