Phospholipid metabolites have a central role in under-plied to study both PME-phosphocholine (PC) and PEand PDE-glycerophosphocholine (GPC) and glycero-standing membrane metabolism in health and disease. In this Communication, we present a new method that makes their phosphoethanolamine (GPE) -in tissue and cell extracts. This is because the PC, PE, GPC, and GPE resonances can quantification possible by 1 H NMR, instead of 31 P NMR or chromatographic methods. The time needed for the 1 H NMR be well resolved in 31 P spectra (17). In other respects, however, 31 P is far from an optimum nucleus for analytical work. procedure is only about 10% of the time necessary when using 31 P NMR. The method is tested and applied in the The NMR sensitivity of 31 P is only approximately 1/15 the sensitivity of 1 H. Often small tissue biopsies and limited cell case of human and rat brain-tissue extracts, and novel concentrations resulting from the phospholipid metabolites in concentrations together with the low number of resonating nuclei also result in prolonged experiments, particularly if the case of hippocampi of temporal lobe epilepsy patients are presented.
absolute quantification is required. The nuclear Overhauser effect in decoupled experiments and the very long T 1 relax-NMR spectroscopy is widely used to investigate cell and tissue biochemistry (1-3). System heterogeneity as well as ation times of the metabolite resonances in 31 P NMR are further disadvantages that complicate quantitative spectros-experimental difficulties limits the information available in vivo. Better characterized in vitro preparations are thus often copy.
It would thus be beneficial to be able to use 1 H NMR utilized to obtain more specific metabolite information (4, 5). Membrane metabolism is one of the key areas in spectroscopy where fully quantitative conditions are quite straightforward to establish. Additionally, in a single 1 H understanding biological phenomena. The biochemical processes involved are complex, but are known to be closely NMR experiment, both the phospholipid metabolites and a related to phospholipid metabolites, phosphomonoesters large number of other important metabolites can be detected. (PME), and phosphodiesters (PDE), which may be used as The reason the efficient use of the proton nucleus in the indicators of cell proliferation, growth, and transformation quantification of the phospholipid metabolites has been pre-(6, 7). Several studies have been undertaken to define the vented is heavy overlap of the resonances. However, as redetailed role of the phospholipid metabolites under different cently illustrated in other biomedical NMR spectroscopy appathological conditions such as tumors and neurological displications (18-26), the use of proper biochemical prior orders . For instance, it has been suggested that phosknowledge together with the application of sophisticated phoethanolamine (PE) may be related to increased seizure lineshape-fitting analysis can overcome this difficulty and activity (15). In all, these studies have revealed the imporyield efficient and reliable quantification of very complex tance of the accurate measurement of individual phospho-NMR data. Here we describe a new method that introduces lipid metabolites in order to understand their biochemical and incorporates biochemical prior knowledge into the specand cellular functions.
tral analysis and makes it possible to quantify the phospho-Chromatography can be used for phospholipid-metabolite lipid metabolites by 1 H NMR. quantification but the methods are often laborious and selec-Biopsy specimens from the hippocampi of epilepsy pative; i.e., different types of protocols and columns are needed tients undergoing temporal lobectomy were frozen in liquid to separate and measure the different metabolites . nitrogen and extracted with perchloric acid (12% w/w Therefore, 31 P NMR spectroscopy has commonly been ap-PCA). Specimens were homogenized and centrifuged at 18,000 g for 20 min at 4ΠC. Pellets were reextracted with PCA and centrifuged as above. Pooled supernatants were