Optimizing blood microsampling protocols for metabolomics and lipidomics: Application in a rehabilitomics study

Aim of the study: Blood microsampling is a promising tool for blood collection as it is simple, minimally invasive and can be configured as a point-of-care device. It is a suitable alternative to venous blood collection and promotes the shift to subject-centered approaches. It enables remote and multiple sampling and is optimal for longitudinal studies1. Metabolomics is a useful tool to investigate metabolic changes induced by physical exercise, as metabolic adaptations occur in a short period of time2. Moreover, a correlation between metabolism and cardiovascular risk has been demonstrated3. The aim of this project is to optimize the extraction protocol for metabolomics and lipidomics analysis of dried blood spots (DBS). The short-term stability of DBS was investigated at room temperature (RT) before storage at -80°C is required. The optimized protocol was then applied to a rehabilitomics study involving patients affected by myocardial infarction participating in a physical rehabilitation program to evaluate exercise-induced changes, correlation with clinical outcome, and cardiovascular risk factors. Materials and methods: Five different extraction solutions were tested using three blood microsampling devices: Whatman, Capitainer B and Telimmune. To investigate short-term stability, samples were left at RT for 0, 1, 2, 3 and 4 days before being stored at -80°C. Blood was collected with DBS at 7 time points: before the start of the rehabilitation protocol, before and after the first training, before and after training halfway through the rehabilitation protocol, and before and after the last training. Untargeted metabolomics analysis was performed using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Results: The best extraction solutions were methanol 80% for metabolomics and methanol 100% for lipidomics as well as metabolomics and lipidomics on the same spot. Polar metabolites were found to be stable at RT for up to 5 days without significant changes. Lipids were stable at RT in Capitainer B for up to 5 days, while some significant changes were observed in Whatman 903 and Telimmune after 2 days. The preliminary rehabilitomics results showed that the most altered metabolic pathways were associated with purine and histidine metabolism. In particular, xanthine and histidine levels were high at the beginning of rehabilitation and gradually decreased with the training time. Discussion and conclusions: An optimized extraction protocol for metabolomics and lipidomics was established and the short-term stability of DBS and DPS was assessed. Overall, the use of DBS simplified longitudinal blood sampling and allowed the investigation of metabolic changes during the physical rehabilitation program after myocardial infarction.