A variable-temperature, -pressure, and -ionic strength 1 H NMR study of the DOTA complexes of different trivalent cations (Sc, Y, La, Ce f Lu) (DOTA ) 1,4,7,10-tetraaza-1,4,7,10-tetrakis(carboxymethyl)cyclododecane) yielded data that are in contradiction with the hitherto used model of only two enantiomeric pairs of diastereoisomers that differ in the ligand conformations. A two-isomer equilibrium cannot explain the newly observed apparent reversal of the isomer ratio at the end of the series. As both conformers may lose their inner sphere water molecule, a coordination equilibrium may be superimposed on this conformational equilibrium, as shown by large positive reaction volumes for the isomerization of [Ln(DOTA)(
PurposeTo differentiate the plasma metabolomic profile of patients with age related macular degeneration (AMD) from that of controls, by Nuclear Magnetic Resonance (NMR) spectroscopy.MethodsTwo cohorts (total of 396 subjects) representative of central Portugal and Boston, USA phenotypes were studied. For each cohort, subjects were grouped according to AMD stage (early, intermediate and late). Multivariate analysis of plasma NMR spectra was performed, followed by signal integration and univariate analysis.ResultsSmall changes were detected in the levels of some amino acids, organic acids, dimethyl sulfone and specific lipid moieties, thus providing some biochemical information on the disease. The possible confounding effects of gender, smoking history and age were assessed in each cohort and found to be minimal when compared to that of the disease. A similar observation was noted in relation to age-related comorbidities. Furthermore, partially distinct putative AMD metabolite fingerprints were noted for the two cohorts studied, reflecting the importance of nutritional and other lifestyle habits in determining AMD metabolic response and potential biomarker fingerprints. Notably, some of the metabolite changes detected were noted as potentially differentiating controls from patients diagnosed with early AMD.ConclusionFor the first time, this study showed metabolite changes in the plasma of patients with AMD as compared to controls, using NMR. Geographical origins were seen to affect AMD patients´ metabolic profile and some metabolites were found to be valuable in potentially differentiating controls from early stage AMD patients. Metabolomics has the potential of identifying biomarkers for AMD, and further work in this area is warranted.
The aim of this work was to investigate the effects of cell handling and storage on cell integrity and 1 H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was ∼70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation-and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The 1 H HRMAS spectra of the three cell types are compared and discussed.Nuclear magnetic resonance (NMR) spectroscopy has been, in recent years, increasingly employed for the analysis of metabolic processes in biological systems because of its ability to provide rapid detection of many different metabolites present in complex systems such as biofluids, biological tissues, or cells. The analysis of the metabolome of biological systems provides important information on their biochemical phenotypes and on the metabolic changes occurring in response to external stimuli, e.g., drug exposure, disease onset, medication. 1,2The study of cellular metabolism using NMR has been successfully carried out with strong emphasis on cell extracts, either hydrophilic or lipophilic. For instance, acidic extracts, in the presence of ice-cold perchloric acid (PCA) or trichloroacetic acid (TCA), allow polar metabolites to be identified 3,4 as shown for PCA extracts of human colon adenocarcinoma cells 5 and human osteosarcoma cells 6,7 and TCA extracts of human rhabdomyosarcoma cells 8 and human lung cancer cells. 9 Other extraction methods have been used to identify aqueous and lipophilic metabolites, for instance in human colon carcinoma cells, 10 rat astrocyte cells, 11 human prostate cancer cells, 12 and human lung carcinoma cell lines. 13 In addition to the unavoidable selectivity of extraction methods, rendered useful only when the nature of the compounds of interest is known a priori, sample extraction may involve significant loss of particular cellular components, retained in the residual insoluble precipitate and not amenable to study by solution-st...
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