# Stability of Nε-Carboxymethyllysine and Nε-Carboxyethyllysine in Canine Urine Under Extended Room Temperature Storage

## TL;DR

This study shows that certain compounds linked to chronic diseases remain stable in dog urine when stored at room temperature for up to a week before freezing.

## Contribution

The study demonstrates that delayed freezing does not significantly affect the measurement of AGEs in canine urine.

## Key findings

- CML and CEL concentrations in dog urine remained stable during room temperature storage for up to 168 hours.
- No significant association was found between storage duration and analyte concentration.
- Inter-replicate agreement was high, supporting the reliability of measurements under these conditions.

## Abstract

Advanced glycation end products are compounds that form in the body and in food during heating and processing. They have been linked to chronic diseases in people and may also play a role in dog health. Measuring these compounds in urine is appealing because urine collection is non-invasive and useful for nutrition and health studies. However, urine samples are not always frozen immediately after collection, and it is unclear whether delays in processing affect the accuracy of these measurements. In this study, urine was collected from healthy dogs and stored at room temperature for up to seven days before freezing. Two advanced glycation end products were measured using a highly sensitive laboratory method. The results showed that concentrations of these compounds remained stable over time, even when freezing was delayed. This suggests that short-term room temperature storage does not substantially affect their measurement in dog urine. These findings support the use of urine samples collected under real-world conditions and provide practical guidance for researchers conducting nutrition, metabolism, and health studies in dogs.

Advanced glycation end products (AGEs) such as Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) are implicated in chronic disease processes in humans and may serve as biomarkers of dietary exposure and metabolic health. Urinary measurement of AGEs is of interest due to its non-invasive nature and relevance to biobanking and field-based sample collection; however, AGE stability in urine under common handling conditions has been poorly characterized. This study evaluates the short-term stability of CML and CEL in canine urine stored at room temperature (20 °C) for up to 168 h prior to −80 °C storage. Midstream free-catch urine samples from eight healthy dogs were aliquoted, stored at defined intervals, and analyzed in duplicate using liquid chromatography–mass spectrometry (LC-MS) with isotope-labeled standards. Results demonstrate minimal detectable changes in CML and CEL concentrations, as well as in the CML/CEL ratio, over the ambient storage period. Inter-replicate agreement is high, and regression and non-parametric analyses show no association between storage duration and analyte concentration. These findings indicate that urinary CML and CEL measurements may remain reliable despite delayed processing, supporting field-based sampling and retrospective analyses. Evaluation of additional AGE species and storage conditions will further inform best practices for sample handling in veterinary and comparative biomedical research.