De novo synthesis of phospholipids and sphingomyelin in multipotent stromal cells - Monitoring studies by mass spectrometry

Musculoskeletal diseases are extremely widespread and a significant burden on the health systems of the industrialized countries. The use of mesenchymal stromal cells is a promising approach to cure cartilage and tendon injuries, which often also occur in younger people as consequences of sport accidents. Although particular interest is on the collagen and the glycosaminoglycan composition of the tendon and potential alterations compared to healthy tissue, there is nowadays also increasing evidence that some selected phospholipids (PL) are potential mediators of tissue regeneration. Therefore, PL (and potential changes thereof) attract increasing interest in this field. We have used positive and negative ion matrix-assisted laser desorption/ionization time-off-light (MALDI-TOF) mass spectrometry (MS) to elucidate the lipid compositions of human mesenchymal stromal cells in dependence on the composition of the cell culture medium and the cultivation time. The de novo biosynthesis of PL was monitored by adding C-13 labeled glucose or deuterated palmitic acid (d(31)-PA) to the cells and the incorporation of C-13 or H-2 into the different PL classes was investigated by electrospray ionization (ESI) mass spectrometry (MS). It is remarkable that all PL classes (for instance, phosphatidylcholine and -inositol) exhibited C-13 incorporation - but not the sphingomyelin (SM) which is the most abundant sphingolipid in the majority of human tissues and body fluids. Using suitable internal standards it could be shown, that only C-12-containing SM is de novo generated while no C-13-labeled SM could be monitored - independent of the cultivation time, which was varied between 7 and 28 days. SM impurities stemming from the cell culture medium and the used MALDI matrix compounds (2,5-dihydroxybenzoic acid (DHB) or 9-aminoacridine (9-AA)) could be ruled out. However, incorporation of deuterated palmitic acid (d(31)-PA) could be observed for multiple PL, including SM. Therefore, it is suggested that there must exist another, so far unknown SM biosynthesis pathway. This pathway does not make use of glucose but relies on the use of other molecules as energy sources. Potential pathways to explain the experimental observations are discussed.