65℃ Short-Term Heating: A Method for Transporting Urine Samples Over Long Distances Without Cold Chain

Biomarkers are measurable changes associated with physiological or pathophysiological processes in the body. Urine, not strictly regulated by homeostatic mechanisms, accumulates extensive variable information and reflects changes in the body earlier and more sensitively, making it an excellent source for biomarker discovery. However, long-distance transport of urine samples usually requires a strictly maintained cold chain throughout the process, resulting in high costs and operational challenges. Here, we propose a urine sample processing method that uses brief heating to inhibit bacterial proliferation and protein degradation. A single urine sample was equally divided into three groups: a pre-heated transport group, a non-heated transport group, and a -80{degrees}C frozen storage control group, with five replicates in each group. Both the pre-heated and non-heated transport groups were transported simultaneously across northern and southern China for five days under ambient temperature conditions. Samples were analyzed using liquid chromatography coupled with tandem mass spectrometry. The results showed that the intra-group overlap rates of identified proteins within the groups treated with 65{degrees}C water bath heating for 15 minutes and -80{degrees}C frozen storage were 97.1% and 97.5%, respectively, demonstrating high reproducibility. There were no significant differences in the number or types of urinary proteins identified between these two groups, with an inter-group overlap rate of 99.9%. Additionally, the pre-heated transport group and frozen storage control group showed no significant difference in the number of bacterial-derived peptides and proteins identified. In contrast, significantly more bacterial-derived peptides and proteins were identified in the non-heated transport group than in both the pre-heated transport and frozen storage control groups. This indicates that heating effectively suppresses bacterial counts in urine samples during five days of transport at ambient temperatures. This approach provides a more economical and convenient solution for long-distance transport of urine samples, eliminating the need for cold chain transportation or preservatives. We also recommend using consistent urine processing methods within the same study to minimize technical variability.