Mass-spectrometric analysis of glycerophospholipid metabolism

This thesis consists of five parts. In the first part, an automated method for quantitative analysis of phospholipid compositions of cells and tissues by liquid chromatographymass spectrometry was developed. In the second part, this method was applied to investigate brain lipid compositions of patients with progressive epilepsy with mental retardation (EPMR), caused by mutations in the CLN8 gene. We were able to show major progressive alterations in brain lipid profiles of EPMR patients which may contribute to disease pathogenesis in those patients. In the third part, a novel approach to investigate the metabolism of single glycerophospholipid molecular species in living cells was developed. This approach was applied to study mechanisms of acyl chain remodeling, i.e. the exchange of fatty acyl residues, of aminophospholipids in BHK and HeLa cells. In the fourth part a novel mass-spectrometric approach was developed to investigate the substrate specificity of phospholipases and was utilized to elucidate the specificities of secretory A-type phospholipases in unprecedented detail. We showed that the specificity of those phospholipases depended mainly on the propensity of the substrates to efflux from the membrane and interactions between the substrate and the enzyme catalytic site are secondary. In the fifth part of this thesis, mechanisms of mammalian glycerophospholipid homeostasis were reviewed and novel theoretical considerations presented.