Figure 1.
Representative NMR spectra of native LDL, oxLDL and eLDL.
(Bottom) Native LDL gave rise to lipid signals predominantly of methyl and methylene protons of fatty acid chains. (Center) NMR spectra of oxLDL showed substantially decreased lipid signals compared to native LDL. (Top) eLDL spectrum: resonances of lipids were virtually absent. Instead, amino acid signals and mobile protein peaks were observed. NMR spectral parameters: 800 MHz spectrometer frequency, 1D-NOESY pulse sequence with water presaturation (noesygppr1d), 7.7 s repetition time, 10 ms mixing time, temperature: 25°C. Spectral annotation according to dominant contributions to NMR signals. Spectra were normalized to the concentration of apoB-100 determined prior to oxidation or modification. Each LDL subtype was investigated on the basis of at least three replicates from different donors, N = 3 except for “oxLDL” (N = 2).
Figure 2.
Changes in NMR-visible lipids as consequence of loading macrophages with modified LDL.
(A) Moderate mobile lipid signals were present in NMR spectra of native macrophages (control). Loading with native LDL or oxLDL did not result in any significant increase of mobile lipid intensities, whereas eLDL loading gave rise to a dominant lipid signal increase. NMR spectral parameters: 800 MHz spectrometer frequency, gradient-based water suppression pulse sequence (zgesgp) with additional water presaturation, 3.7 s repetition time, temperature: 5°C. Spectra were normalized to protein and/or glutathione (GSH) signal intensities. Spectral annotation according to dominant contributions to NMR signals. (B) The NMR-visible lipid content, i.e. the integral in arbitrary units over the deconvolved NMR signal of the terminal methyl group of fatty acid chains. (C) The average percentage of bis-allylic methylene per fatty acid chain in control macrophages and macrophages loaded with native LDL, oxLDL and eLDL, i.e. the ratio of NMR-visible bis-allylic methylene groups and methyl groups. Deconvolved peaks of (-CH = CH-CH2-CH = CH-)- and (-CH3)-protons were integrated. Mean and standard deviation of five samples from different donors, N = 5 except for “LDL” (N = 4). Mann-Whitney U test: **p<0.01.
Figure 3.
Effects of modified LDL on lipid extractions of macrophages.
(A) NMR spectra of extracted lipids. “Blank”: Extracted components of the sample cup (This spectrum is subtracted from cell extract spectra prior to quantification). Shaded regions were used for quantification. NMR spectral parameters: 800 MHz spectrometer frequency, pulse-acquire pulse sequence (zg), 14.6 s repetition time, temperature: 5°C. Spectral annotation according to dominant contributions to NMR signals. (B) The total intracellular lipid content, i.e. the in arbitrary units over the spectral region between 0.8 ppm and 0.95 ppm dominated by protons of the terminal methyl group of fatty acid chains. (C) The average percentage of bis-allylic methylene per fatty acid chain in control macrophages and macrophages loaded with native LDL, oxLDL and eLDL, i.e. the ratio of bis-allylic methylene groups and methyl groups. Spectral regions of (-CH = CH-CH2-CH = CH-)- and (-CH3)-protons were integrated (2.75–2.9 ppm and 0.8–0.95 ppm, respectively). Mean and standard deviation of three samples from different donors except for “LDL” (N = 2).
Figure 4.
ESI-MS/MS analysis of lipoprotein loaded macrophages and native LDL, oxLDL and eLDL.
(A) eLDL induced significant accumulation of mono- and polyunsaturated lipid species in macrophages. oxLDL only elevated levels of monounsaturated species. (B) Lipid compositional analysis of lipoproteins. oxLDL showed substantially decreased levels of mono- and polyunsaturated lipids compared to native LDL. Saturated lipid species were decreased in eLDL, but increased in oxLDL. Mean and standard deviation of six (A) or eleven (B) replicates from different donors. Mann-Whitney U test: *p<0.05, **p<0.01.