Isolation und Identifikation von Lipiden in Körperflüssigkeiten.

Abstract

The analysis of intact and underivatised lipids is one of the challenging tasks of body fluids as well as cell and tissue extracts. The lipid composition consists of different classes such as neutral and positively or negatively charged lipids with manifold subclasses of structural diversity. Phospholipids consist of a polar head group ( i.e. phosphorylcholine, etc.) attached to the sn-3 position of glycerol and varying saturated and unsaturated fatty acids esterified to the sn-1 and sn-2 position whereby fatty acids in position sn-1 were mostly saturated. Structural diversity will have a distinct effect on membrane properties (i.e. fluidity, permeability, oxygen scavenger, etc.). Anomalous membrane compositions were correlated to neoplastic and neurodegenerative diseases, diabetes and many others. Further more they are involved in cell death (apoptosis, necrosis), cellular signaling and are precursers for lysophosphatidylcholines, diacylglycerols, phosphatic and arachidonic acid. Many different analytical techniques have been proposed to characterize pathophysiological deviations of the native lipid composition, but most of them are destructive. Further more most of these studies recorded only the total amount of the individual lipid classes without acquiring additional information on the composition of structural subclasses. With NMR spectroscopy it is possible to measure intact biomaterials nondestructively without any preceding derivatisation, but its low sensitivity limits a wide application. To overcome the limitations of a single analytical tool the combined approach in lipid analysis should improve the reliability of the results and eventually will reveal yet unknown biomarkers or species. Therefore, an efficient analytical tool is needed to identify and quantify in situ low concentrated known, but also still unidentified naturally occurring species to get a quick insight into biochemical processes. The combination of HPLC separation power, MS sensitivity with accurate mass measurement of molecular and fragment ions and NMR structure elucidation power will meet most suitably the challenge and will overcome the limits any single technique, but also demonstrate the potential of their combination ultimately to analyze native lipid mixtures. By means of the HPLC separation with novel isocratic reversed phase HPLC-methods (separation according to the fatty acid composition) it was possible to get semi quantitative information of the individual species in the phosphatidylcholin, phosphatidylethanolamin, phosphatidylserine, phosphatidylinositol and sphingomyeline extracts. MS peak detection offers a high sensitivity to detect low concentrated compounds but has only a low specificity. MS/MS experiments overcome this problem and allow the assignment of individual fatty acids to the sn-1 or sn-2 position within the glycerol moiety. By high resolution spectra it is additional possible to generate the compound's molecular formula to detect abnormalities like oxidation of the double bond etc. The separated phospholipids were pooled and sampled in a 600 MHz NMR-spectrometer for recording 1D and 2D spectra to assess the potential, power and universality of this method. Based on the MR-data (1H, 13C or 31P) the lipid class, degree of unsaturation and anomalies were easily identified. The hyphenated approach benefits most by the NMR spectroscopy structure elucidation power in complex mixtures. In routine analysis data base identification by retention time, precision mass and 1H NMR helps to save money and time. Only new, abnormal or unclear sample compounds should be selected for more time consuming 2D NMR techniques. Detailed experimental features and results obtained on these lipids are given in the document.