Urine stability for metabolomic studies: effects of preparation and storage

Abstract

Metabolomic studies attempt to identify and profile unique metabolic differences among test populations, which may be correlated with a specific biological stress or pathophysiology. Due to the ease of collection and the metabolite-rich nature of urine, it is frequently used as a bio-fluid for human and animal metabolic studies. High-resolution 1 H-NMR is an analytical tool used to qualitatively and quantitatively identify metabolites in urine. Urine samples were collected from healthy male and female subjects and prepared: raw, following centrifugation, filtration, or the addition of the bacteriostatic preservative sodium azide and analyzed by NMR. In addition, these samples were stored at room temperature (22°C), in a refrigerator (4°C), or in a deep-freeze ()80°C). Samples were analyzed by NMR every week for a month and changes in concentrations of 55 easily identifiable metabolites were followed. The degree of change in metabolite concentrations following storage over a 4-week period were influenced by the different methods of sample preparation and storage. Significant changes in urine metabolites are likely due to bacterial contamination of the urine. Our study demonstrates that bacterial contamination of urine in normal individuals significantly alters the metabolic profile of urine over time and proper preparation and storage procedures must be followed to reduce these changes. By identifying appropriate methods of urine preparation and storage investigators will preserve the fidelity of the urine samples in order to better reflect the original metabolic state.

A comparison of metabolite extraction strategies for 1H‐NMR‐based metabolic profiling using mature leaf tissue from the model plant Arabidopsis thaliana

“…In any case where aqueous or part-aqueous solvents were employed, a 100 mM deuterated ammonium acetate buffer was introduced to reduce variance in the chemical shifts of pH-sensitive metabolites, as recommended by Colquhoun. [30] Additionally, 0.5 mM sodium azide was added as a bacteriostat [31,32] and 100 µM trimethylsilylpropionate-d 4 (TMSP-d 4 ) was utilized as a chemical shift reference. Following perchloric acid treatment (method 5), extracts were titrated to a pH meter reading of 4.76.…”

Method Validation for Preparing Serum and Plasma Samples from Human Blood for Downstream Proteomic, Metabolomic, and Circulating Nucleic Acid-Based Applications

“…Where proteomic and metabolomic research is concerned, pre-processing and processing of samples at 4°C is considered essential. Not only does it minimize protein degradation due to protease activity, [24][25][26] it also limits metabolite changes that can occur due to bacterial growth at RT, 27 and potential microbial growth due to the nonsterile nature of urine. 28 Glycine could be unambiguously identified and was found to be poorly reproducible.…”

NMR metabolomics and drug discovery

“…[50] Specifically, issues such as long-term storage, [62,63] protein removal, [46] selection of extraction solvent, [45] and tissue preparation [47] can all affect the quality and reliability of the analysis. The advantage of PCA is the extreme sensitivity of the method to subtle spectral differences.…”

“…[86] Fundamental variabilities in an individual's metabolome resulting from age, gender, genetics, environmental exposure, behavior, or diet differences may mask the impact of a disease or incorrectly imply the presence of a disease. Other factors, such as the collection, storage, and handling of the biological samples [62,63] or measurement errors, [51,64] may also compromise the correct identification and utility of biomarkers.…”