Abstract
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.
Highlights
At relatively small rotational correlation times all molecules can be detected in solution via 1H NMR spectroscopy
Such an effect is clearly observed for lipoproteins that are well visible in 1H NMR spectra
We investigated 1) what proportion of the expected lipid signals for different lipids in different lipoproteinclasses can be observed at different temperatures by 1H NMR spectroscopy; and 2) characterized these effects by a thermodynamic analysis of the temperature-dependent changes of spectral shapes and intensities
Summary
Lipoprotein particles of different size and composition mediate lipid metabolism by transporting them to their place of destination via the blood or the lymphatic system [1, 2]. The outer layer of all lipoprotein classes is covered by hydrophilic moieties of apolipoproteins and of amphiphilic lipids, such as free unesterified cholesterol, phospholipids (PLs), and SM, in order to guarantee water solubility of the assembly. The lipoprotein composition directly affects the molecular dynamics of the lipids and may alter the conformation of apolipoproteins, which has direct influence on metabolic processes like receptor binding or cholesterol exchange [3,4,5]. Lipid assemblies usually are subjected to a phase transition from a solid-like gel state to a liquid-crystalline-like state with higher internal mobility. The core lipids of LDLs undergo a broad phase transition
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