NMR-based Lipoprotein Subclass Analysis

Serum levels of apoB-containing lipoproteins were measured on a Bruker 600 MHz Avance Neo NMR spectrometer using the Bruker IVDr lipoprotein subclass analysis protocol, as described previously [7 (link),48 (link)]. Briefly, serum samples were thawed, and 330 µL of each sample was mixed with 330 µL of Bruker serum buffer (Bruker, Rheinstetten, Germany). The samples were mixed gently and 600 µL of the mixed sample was transferred into a 5 mm SampleJet rack tube (Bruker, Rheinstetten, Germany). Proton spectra were obtained at a constant temperature of 310 K using a standard Nuclear Overhauser Effect Spectroscopy (NOESY) pulse sequence (Bruker: noesygppr1d), a Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence with presaturation during the relaxation delay (Bruker: cpmgpr1d) to achieve water suppression, and a standard 2D J-resolved (JRES) pulse sequence (Bruker: jresgpprqf). Data analysis was carried out using the Bruker IVDr Lipoprotein Subclass Analysis (B.I.LISA™, Bruker, Rheinstetten, Germany) method. Lipid contents of apoB-containing lipoprotein particles were calculated as ratios of serum levels of the respective lipid in apoB-containing lipoprotein (mg/dL) and apoB in apoB-containing lipoprotein (mg/dL).