Urine Sample Collection and Preparation in Metabolomics

Urine is one of the most widely used biological samples in metabolomics research, with characteristics such as non-destructive and reproducible collection. Urine contains a large number of metabolic end products and carries a wealth of metabolic information.

The main component of urine is water, accounting for 96% to 97%, while others are urea, uric acid, creatinine, ammonia, sulfate, etc. The wide pH range, concentration, and ionic strength of urine pose certain challenges for metabolomic analysis, and corresponding sample preparation and technical analysis methods need to be established.

Urine Collection and Storage

In animal experiments, the use of metabolic cages has greatly facilitated the collection of urine. However, there are still many details that need attention during the sample collection process, such as the stress to animals by rash use of metabolic cages; contamination of urine with food residues and feces; evaporation of urine and contamination of urine collection bottles with plasticizers.

Urine collection should give due consideration to preservatives, drinking water, food, and time of collection. Sodium azide (NaN₃) is a commonly used preservative. It is added to urine collection bottles (0.05% to 1%) to inhibit microbial contamination and the degradation of its resulting products. However, there are studies confirming no significant differences in metabolic profiles and principal component data analysis with or without the addition of sodium azide (3 mmol/L). The analysis suggests that there may be relevant differences between samples from different treatments, but the method of analysis of principal components may mask the corresponding test results.

In the experimental design, the animals should be fasted and dehydrated according to the specific requirements of the test purpose, and the time of urine collection should be given due consideration. Fasting and other measures may lead to changes in urine volume, composition and concentration. For samples with chronic renal failure, hyperlipidemia and other diseases, the effect of water or food fasting on urine collection should be considered.

Rodents are more active at night. Urine output is large and informative about metabolites. Daytime collections may have low or no urine output. Urine collected at different times of the day has a large variation in metabolic profile.

Prolonged collection times (>12h) can result in prolonged exposure of urine, which can lead to evaporation, contamination and product degradation. Therefore, it is recommended that the collection tube be placed in ice or dry ice to control the temperature.

The urine is collected and centrifuged promptly at low temperature and high speed (4°C) to remove cells, particles and other impurities. The time interval between sample collection and centrifugation should not exceed 2 h. The centrifuged samples should be stored in a low temperature refrigerator. Short-term storage at -20°C is possible. Longer periods need to be stored at -80°C or in liquid nitrogen. Avoid repeated freezing and thawing of samples. It is recommended that samples be stored for no longer than 26 weeks.

Urine Sample Preparation

The urine pretreatment methods are divided into 2 main types as follows:

1. Mixing urine and water in a certain ratio (1:1 to 1:4). After mixing, centrifuge at low temperature and high speed, and take the supernatant to filter the assay.

2. The urine is shaken with a proportion of organic solvent (methanol or acetonitrile). The supernatant is then filtered and assayed by centrifugation at low temperature and high speed to remove the protein.

Human urine is low in protein and usually does not require deproteinization. In rodents, physiological urine protein is present and de-proteinization is recommended. The supernatant samples obtained by both methods are filtered through a membrane. The pore size of the membrane is typically 0.22 pm.

Affinity chromatography and solid-phase extraction techniques are suitable for the pre-treatment of urine when extracting certain components of specific nature in urine. In specific experiments, the urine treatment method should be selected and optimized in combination with the instrument, mobile phase, analytical column characteristics and pre-test results. For example, the choice of organic reagent methanol or acetonitrile, the ratio of urine to water or organic solvent, centrifugation speed, centrifugation time, etc.

Workflow of GC–MS-based untargeted metabolomic analysis of urine (Khodadadi et al., 2020)

Reference

1. Khodadadi, M., & Pourfarzam, M. (2020). A review of strategies for untargeted urinary metabolomic analysis using gas chromatography–mass spectrometry. Metabolomics, 16(6), 1-14.