1H NMR spectroscopy quantifies visibility of lipoproteins, subclasses, and lipids at varied temperatures and pressures.

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.

Identification of molecular species of cholesteryl ester hydroperoxides in very low-density and intermediate-density lipoproteins.

BACKGROUND: Oxidation of lipoproteins is thought to play a crucial role in atherogenesis. Role for triglyceride-rich lipoproteins in atherogenesis is unclear. Thus, we aimed to investigate whether cholesteryl ester hydroperoxides (CEOOH) are present in very low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) by using highly sensitive liquid chromatography/mass spectrometry. METHODS: Total lipids were extracted from the plasma of healthy donors (n = 6) and their fractions of VLDL and IDL. Additional three plasma samples were analysed freshly for CEOOH. Detection and identification of CEOOH was conducted by liquid chromatography/LTQ ion trap mass spectrometry/Orbitrap high mass accuracy mass spectrometry. Authentic standards of CEOOH were used for unequivocal identification on the basis of their mass spectra. RESULTS: We identified six molecular CEOOH species overall, namely, Ch18:1-OOH, Ch18:2-OOH, Ch18:3-OOH, Ch20:4-OOH, Ch20:5-OOH and Ch22:6-OOH. Of them, Ch18:2-OOH, Ch20:5-OOH, Ch20:4-OOH and Ch22:6-OOH were detected in all IDL samples, while only Ch20:4-OOH was detected in all VLDL samples. All of CEOOH species except for Ch18:3-OOH were detected in plasma, with constant detection of Ch20:5-OOH, and Ch22:6-OOH in all plasma samples. CONCLUSION: The presence of CEOOH species in VLDL and IDL was confirmed with the analytical sensitivity of 0.1 pmol, showing the constant appearance of more CEOOH species in IDL than VLDL. This finding might add biochemical evidences of atherogenicity of these lipoproteins. Clinical utility of measuring CEOOH level in these lipoproteins need to be investigated for the risk assessment of the cardiovascular disease.