High-Coverage Quantitative Metabolomics of Human Urine: Effects of Freeze–Thaw Cycles on the Urine Metabolome and Biomarker Discovery

Urine sample storage after collection
at ultra-low-temperature
(e.g., −80 °C) is normally required for comparative metabolome
analysis of many samples, and therefore, freeze–thaw cycles
(FTCs) are unavoidable. However, the reported effects of FTCs on the
urine metabolome are controversial. Moreover, there is no report on
the study of how urine FTCs affect biomarker discovery. Herein, we
present our study of the FTC effects on the urine metabolome and biomarker
discovery using a high-coverage quantitative metabolomics platform.
Our study involved two centers located in Hangzhou, China, and Edmonton,
Canada, to perform metabolome analysis of two separate cohorts of
urine samples. The same workflow of sample preparation and dansylation
isotope labeling LC–MS was used for in-depth analysis of the
amine/phenol submetabolome. The analysis of 320 samples from the Hangzhou
cohort consisting of 80 healthy subjects with each urine being subjected
to four FTCs resulted in relative quantification of 3682 metabolites
with 3307 identified or mass-matched. The analysis of 176 samples
from the Edmonton cohort of 44 subjects with four FTCs quantified
3516 metabolites with 3166 identified or mass-matched. Multivariate
and univariate analyses indicated that significant variations (fold
change ≥ 1.5 with q-value ≤ 0.05) from
FTCs were only observed in a very small fraction of the metabolites
(<0.3%). Moreover, various metabolites did not show a consistent
pattern of concentration changes from one to four FTCs, allowing the
use of two separate cohorts of samples to remove these randomly changed
metabolites. Three metabolite biomarkers for separating males and
females were discovered, and FTC did not influence their discovery.