Short communication: do milk purine derivatives reflect the urinary PDC index in dairy cows? Insights from integrative milk–urine targeted and untargeted screening

Urinary purine derivatives (PD) are widely used as non-invasive indicators of microbial protein synthesis in dairy cows, whereas their potential use in milk as an alternative biofluid remains poorly understood. This study investigated whether milk PD reflect urinary purine metabolism, assessed the effects of dietary protein degradability on PD profiles, and explored coordinated metabolic responses in milk and urine using an integrative targeted and untargeted approach. We hypothesised that differences in dietary protein degradability would induce coordinated metabolic changes detectable across both biofluids. Milk and urine samples from nine lactating Holstein cows fed diets differing in rumen-degradable (RDP) to rumen-undegradable (RUP) protein ratio, namely CTR (64:36 ratio), SOL (67:33 ratio), and BY (57:43 ratio), were analysed by targeted and untargeted liquid chromatography coupled with high-resolution mass spectrometry. Targeted quantification of allantoin, uric acid, and creatinine revealed no association between milk PD concentrations and the urinary purine derivatives-to-creatinine (PDC) index, indicating that milk PD do not directly reflect urinary purine metabolism. However, untargeted profiling identified diet-related metabolic differences in both biofluids, with changes involving pathways related to amino acid metabolism, particularly in cows receiving the high-rumen-undegradable-protein diet. Integration of milk and urine datasets using DIABLO further revealed coordinated metabolite patterns across biofluids and dietary groups. Overall, these findings indicate that milk PD alone may not be a suitable proxy for urinary PD-based assessment of microbial protein synthesis. Integrated profiling of multiple biofluids can reveal systemic metabolic responses to dietary protein degradability and provide complementary insights into nitrogen metabolism in dairy cows.