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

Author(s): Chen D, Chan W, Zhao S, Li L, Li L.

Publication: Anal Chem, 2022, Vol. 94, Page 9880-9887

PubMed ID: 35758637 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the metabolome of patient-matched urine specimens collected from healthy volunteers that was then subjected to 1-4 freeze-thaw cycles. Potential effects of freeze-thaw cycling were investigated using urine from two separate patient cohorts that were collected in different countries. Additionally, sex-specific differences in the metabolome of urine specimens were investigated. Urine was collected from 80 healthy volunteers (42 female and 38 male) in Hangzhou, China and 44 healthy volunteers (22 female and 22 male) in Edmonton, Canada and placed in 1.5 mL vials. Urine specimens were subjected to between one and four cycles of freezing and thawing. For each cycle, the urine specimen was frozen at -80°C, thawed at room temperature for 90 min, vortexed, centrifuged at 18,000 g at 4°C for 30 min, and sampled before the remaining sample was refrozen.  Pooled specimens representing 1, 2, 3 and 4 freeze-thaw cycles were created by mixing an aliquot of each specimen. Individual and pooled urine specimens were labelled using the Nova MT 12C-dansyl chloride (Dns-Cl) and 13C-Dns-Cl kits, respectively, before analysis with the ltiMate 3000 UHPLC with an Impact II Quadrupole Time-of-flight (QTOF) mass spectrometer. Peaks were analyzed using the IsoMS Pro software, and metabolites were identified using a three-tier approach where tier 1 consisted of positively identified metabolites, tier 2 represented putatively identified metabolites and tier 3 contained predicted metabolites.  The significance of differences observed among metabolite levels was determined by paired-sample t-test with a threshold false-discovery rate-adjusted q-value of < 0.05 at a particular fold change (≥1.5, 1.4, 1.3, or 1.2) in >75% of same subject pairs.

   Summary of Findings:

   A total of 3682 and 3166 metabolites were detected in urine specimens from the Hangzhou and Edmonton cohorts, respectively. In the Hangzhou and Edmonton cohorts 170 and 193 metabolites, respectively, were positively identified (tier 1) and 647 and 571, respectively, were putatively identified with high confidence (tier 2). Of the 216 metabolites positively identified in at least one cohort, 197 were identified in both cohorts; and of the 793 putatively identified metabolites in at least one cohort, 645 were found in both cohorts.  PCA based on the metabolome showed no obvious clustering of specimens based on the number of freeze-thaw cycles, but some clustering based on patient sex was observed. Importantly, the separation between specimens collected from males and females was more pronounced when the specimens were subjected to the same number of freeze-thaw cycles. Using a threshold of ≥1.5-fold change from matched urine specimens that were only freeze-thaw cycled once, 19, 8 and 21 metabolites were significantly altered by 2, 3, and 4 freeze-thaw cycles, respectively, in the Hangzhou cohort. In contrast, only 0, 3, and 24 metabolites exhibited a ≥1.5 fold difference in levels when urine specimens from the Edmonton cohort were subjected to 2, 3 and 4 freeze-thaw cycles, respectively, were compared to controls that underwent one freeze-thaw cycle.  When the threshold for fold-change was lowered, the number of metabolites that differed significantly as a result of freeze-thaw cycling increased, but only 277 and 146 metabolites displayed a ≥1.2-fold change between specimens freeze-thaw cycled 4 times and 1 times in matched specimens from the Hangzhou and Edmonton cohorts, respectively. Interestingly, no metabolites were consistently affected by freeze-thaw cycling in both cohorts.  Levels of three metabolites (guanidoacetic acid, pantothenic acid, and cadaverine) occurred at higher levels in urine from females than males in specimens from both cohort sites. Importantly, these three markers were found to be differentially expressed in urine from females versus males, regardless of the number of freeze thaw cycles; however, when urine was subjected to different numbers of cycles there were many false positives for additional sex-specific markers.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	LC-TOF-MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles
   Preaquisition	Patient gender	Female Male

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