Abstract
Human urine, which is rich in metabolites, provides valuable approaches for biomarker measurement. Maintaining the stability of metabolites in urine is critical for accurate and reliable research results and subsequent interpretation. In this study, the effect of storage temperature (4, 22, and 40 °C), storage time (24 and 48 h), and use of preservatives (boric acid (BA), thymol) and para-aminobenzoic acid (PABA) on urinary metabolites in the pooled urine samples from 20 participants was systematically investigated using large-scale targeted liquid chromatography tandem mass spectrometry (LC-MS/MS)-based metabolomics. Statistical analysis of 158 reliably detected metabolites showed that metabolites in urine with no preservative remained stable at 4 °C for 24 and 48 h as well as at 22 °C for 24 h, but significant metabolite differences were observed in urine stored at 22 °C for 48 h and at 40 °C. The mere addition of BA caused metabolite changes. Thymol was observed to be effective in maintaining metabolite stability in urine in all the conditions designed, most likely due to the inhibitory effect of thymol on urine microbiota. Our results provide valuable urine preservation guidance during sample storage, which is essential for obtaining reliable, accurate, and reproducible analytical results from urine samples.
Highlights
Metabolomics is a field concerned with the comprehensive monitoring of thousands of metabolites in biological systems for assessing metabolic modifications caused by physiological/environmental factors at the molecular level [1,2,3,4,5]
We systematically investigated the effects of three temperatures (4, 22, and 40 ◦ C), two storage durations (24 and 48 h), and three preservative methods (No preservative (NP), boric acid, and thymol) (Table 1) on the stability of metabolite signatures in urine with or without para-aminobenzoic acid (PABA), a compound that is commonly used as a marker of 24 h urine completeness in dietary biomarker studies [35], using a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based metabolomics approach
For the NP samples, the principal component analysis (PCA) score plots showed that controls (−20 ◦ C) and 4 ◦ C samples were clustered together, indicating that urine metabolites remained stable at 4 ◦ C in both PABA (Figure 1A) and no PABA (Figure 1B) pooled samples, regardless of storage duration (24 or 48 h)
Summary
Metabolomics is a field concerned with the comprehensive monitoring of thousands of metabolites (mass < 2000 Da) in biological systems for assessing metabolic modifications caused by physiological/environmental factors at the molecular level [1,2,3,4,5]. Among various biological matrices used in metabolomics, urine is a non-invasive biofluid that carries abundant water-soluble metabolic waste products. Due to the appealing advantages of non-invasive sample collection, ready availability, and richness of information that urine samples provide, urinary tests are performed widely and extensively for the detection of illegal drug use [7,8], renal function monitoring [9,10], diagnosis [11,12,13], cancer risk assessment [14,15], disease biomarker identification [16,17,18], etc., and provide valuable insights into human health and disease pathogenesis. Metabolite signatures in urine can be used as biomarkers of dietary patterns or intake of specific foods, and may help elucidate mechanistic pathways relating diet to chronic disease risk [19]. Urine plays an important role in metabolomics and metabolism research
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