Urine is an excellent source of multiple metabolites, providing detailed insight into an individual’s health status. It is relatively easy to collect, large volumes are available and standard testing procedures are well established. Furthermore, it is an excellent sample to collect for long-term population studies. But how long can you hold it?
Remer et al. (2014) decided to investigate analyte stability using samples from the Dortmund Nutritional and Anthropometric Longitudinally Designed (DONALD) study urine bank.1 The study has been running since 1985, monitoring child growth and development in healthy individuals between 3 and 18 years old. As part of the ongoing open cohort longitudinal study, 24-hour urine samples were collected and stored preservative-free at -22°C. Initial analysis took place at time of collection before researchers divided samples into aliquots for storage. This step maintained integrity for future testing, as samples underwent only one freeze-thaw cycle.
Remer and co-authors chose samples stored for 12 and 15 years, re-analyzing the stored aliquots to compare results for 21 clinical biochemical parameters with those obtained at initial measurement. They ran the assays according to standard protocols, often with the same instrumentation used at the time of initial sample collection. The laboratory instrumentation, including a Thermo Scientific Dionex 200i/SP ion chromatography system with a Dionex ion Pac AS4A column , had good maintenance and quality-control records, so the researchers could show that the assays had performed consistently for the duration of the longitudinal study.
The research team re-analyzed the samples for organic acids (12 years of storage) and anion and cation levels (15 years of storage). They also assayed creatinine, urea, iodine, citric and uric acid, nitrogen, sodium, potassium, magnesium, ammonium, and bicarbonate levels, in addition to re-assessing osmolality and acid-base balance (all 15 years of storage).
Based on the comparison between levels assayed at each of the two time points (initial collection versus after specified storage time), the researchers obtained good recoveries for all analytes (r = 0.94 – 0.99) except oxalate (73%; r = 0.77). Furthermore, they only encountered significant differences for total titrated organic acids at 12 years. For osmolality, anions, titratable acid and oxalate levels, they found significant differences after 15 years of storage. Otherwise, mean percentage differences over the 12-year and 15-year storage periods were only ±5%.
With such excellent results, the research team suggests that its work is a valuable contribution, boosting the rather limited data on long-term stability of urine samples for historical biochemistry studies. Furthermore, their results show that addition of preservative prior to freezing is not necessary, thus avoiding change in pH. Although their work did not consider more specific protein-based biomarkers that need lower storage temperatures for stability, the authors are confident that the results they present show the validity of using stored urine samples in long-term epidemiological studies.
1. Remer, T. et al (2014) “Long-term urine biobanking: Storage stability of clinical chemical parameters under moderate freezing conditions without use of preservatives,” Clinical Biochemistry, pii: S0009-9120(14)00666-3. doi: 10.1016/j.clinbiochem.2014.09.009.