The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or... Show moreThe metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1 Delta/-mouse mutant exhibiting multiple-morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia. Show less
The small proline-rich (SPRR) proteins are generally known for their involvement in the formation and adaptation of the skin__s barrier. During the cornification process, they are cross-linked... Show moreThe small proline-rich (SPRR) proteins are generally known for their involvement in the formation and adaptation of the skin__s barrier. During the cornification process, they are cross-linked within the cornified cell envelope (CE) and as such they are responsible for the physical and permeability barrier function of our skin. In this thesis, the novel antioxidant properties of the SPRR proteins are described. As part of the CE, these proteins provide a natural antioxidant shield to our skin and act as our first line of defence against reactive oxygen species (ROS). Also during wound healing, SPRR proteins can directly reduce toxic ROS levels. This activity is directly related to their ability to promote cell migration and is essential in order to allow wound closure. A literature-based meta-analysis revealed their up-regulation in various forms of tissue injury, ranging from heart infarction and commensal-induced gut responses to nerve regeneration and burn injury. Apparently, SPRR proteins have a far more widespread role in wound healing and tissue remodelling than their established function in skin cornification. Likely, SPRR proteins provide all tissues with an efficient, finely tuneable antioxidant barrier, specifically adapted to the tissue involved and the damage inflicted. Show less
