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Impact of collection conditions on the metabolite content of human urine samples as analyzed by liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy.

Author(s): Roux A, Thévenot EA, Seguin F, Olivier MF, Junot C

Publication: Metabolomics, 2015, Vol. 11, Page 1095-1105

PubMed ID: 26366133 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of preservation with boric acid and refrigeration versus room temperature storage on bacterial growth and the stability of metabolites in urine. Midstream urine from two healthy women and three healthy men was collected between 9:30 and 10:30 AM into containers without preservative and stored at room temperature (26˚C) or 4˚C for 0, 12, 24, 36, 48, and 72 h. Additional midstream urine from three healthy women and two healthy men was collected into containers with 200 mM boric acid or without preservative and stored at room temperature (23˚C) or 4˚C (non-preserved only) for 0, 12, 24, 48, and 72 h. Following storage, urine was frozen at -80˚C until analysis. Bacterial growth was determined by turbidimetry. Prior to LC-MS analysis, specimens were centrifuged at 3000 rpm for 5 min. Prior to NMR analysis, buffer was added, pH was adjusted to 7.0, and urine was centrifuged at an unspecified speed to remove cellular fragments and sediment.

    

   Summary of Findings:

   Bacterial growth as determined by optical density increased drastically in urine stored at room temperature with an approximately 3-fold increase found by 12-24 h but no increase in optical density occurred in boric-acid preserved urine or in urine stored at 4˚C.

   There was some spreading between replicates in the LC-MS unsupervised principle component analysis but specimens segregated into three groups: 1) those stored unpreserved at room temperature, 2) those stored with boric acid at room temperature and 3) those stored unpreserved at 4˚C. There were time-course effects of storage of unpreserved and preserved urine at room temperature that were correlated with the first bisector in the LC-MS partial least-square analysis (PLS) of data from the second experimental group, but these were not observed in the 4˚C stored urine. A total of 184 and 232 metabolites were identified by NMR in the first and second experiments, respectively and 136 of these were common to both experiments. Levels of nineteen metabolites were found to correlate with room temperature and with similar statistical significance between the room temperature and 4˚C stored specimens in both experimental groups. Among the identified metabolites with significant positive correlations to room temperature storage duration in both experiments were creatinine (r=0.99 and r=0.7), cholic acid (r=0.81 and r=0.77), valine (r=0.81 and r=0.71), aniline isomer (r=0.71 and r=0.69), threonolactone (r=0.79 and r=0.94), and orotic acid (r=0.66 and r=0.88) but threonolactone levels were significantly correlated with storage duration at 4˚C in the second experiment only (r=0.91). Significant negative correlations with storage duration at room temperature in both experiments were found for trimethylamine oxide (r=-0.98 and r=-0.92), 2-hydroxy-3-methylbutyric acid (r=-0.98 and r=-0.84), methylguanosine (r=-0.96 and r=-0.81), methylinosine (r=-0.93 and r=-0.88),  glutamine (r=-0.91 and r=-0.88), dimethyguanosine (r=-0.86 and r=-0.71),  3-methyl-2-Oxovaleric acid (r=-0.77 and r=-0.84), N-acetylcytidine (too rapid to calculate and r=-0.83 to -0.91), urobilinogen (r=-0.93 to -0.99 and r=-0.97 to -0.98), urobilin (r=-0.99 and r=-0.98), ketoretinoic acid glucuronide isomer (r=-0.94 and r=-0.98), hydroxyretinoic acid glucuronide isomer (r=-0.91 and r=-0.95), and ascorbic acid (r=-0.88 and r=-0.96). Significant negative correlation with storage duration at 4˚C were observed in both experiments for urobilinogen (r=-0.81 and r=-0.93 to -0.96), urobilin (r=-0.75 and r=-0.89), and ascorbic acid (r=-0.76 and r=-0.91); only in the first experiment for N-acetylcytidine (r=-0.88 to -0.92); and only in the second experiment for ketoretinoic acid glucuronide isomer (r=-0.94) and hydroxyretinoic acid glucuronide isomer (r=-0.86). Duration of room temperature storage of boric acid preserved specimens was negatively correlated with urobilinogen (r=-0.93 to -0.95), urobilin (r=-0.91), ketoretinoic acid glucuronide isomer (r=-0.97), hydroxyretinoic acid glucuronide isomer (r=-0.98), and ascorbic acid (r=-0.98) and positively correlated with threonolactone (r=0.98) and orotic acid (r=0.72).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Small molecule	NMR
   Cell count/volume	Light scattering
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 h 12 h 24 h 36 h 48 h 72 h
   Biospecimen Preservation	Type of fixation/preservation	Boric acid Refrigeration Frozen
   Storage	Storage temperature	4˚C 23˚C 26˚C

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