Metabolomic Characterisation of Low-Density Lipoproteins Isolated from Iodixanol and KBr-Based Density Gradient Ultracentrifugation

Background/Objectives: Salt-based density gradient ultracentrifugation (SBUC) is frequently used to isolate lipoproteins for their subsequent analysis. However, the addition of salts may disrupt their molecular composition. Therefore, the aim of the present study was to assess the impact of SBUC upon the molecular composition of low-density lipoprotein (LDL) particles, compared to a validated non-salt method involving iodixanol gradient ultracentrifugation (IGUC). Methods: Whole human plasma was analysed for various lipid parameters before LDL particles were isolated using both SBUC and IGUC methods. Each fraction was then filtered to obtain low-molecular-weight compounds. The LDL molecular content of the resulting fractions from both methods was determined using untargeted liquid chromatography–mass spectrometry (LC-MS) in positive and negative modes. Results: A total of 1041 and 401 features were putatively identified using positive and negative modes, respectively. Differences were shown in the molecular composition of LDL prepared using SBUC and IGUC; in positive mode ionisation, the PLS-DA model showed reasonable fit and discriminatory power (R2 = 0.63, Q2 = 0.58, accuracy 0.88) and permutation testing was significant (<i>p</i> < 0.001). Conclusions: The findings reveal distinct differences in the small molecule composition of LDL prepared using the two methods, with IGUC exhibiting greater variation. In negative mode, both methods detected phospholipids, long-chain sphingolipids, and ceramides, but IGUC showed higher fold differences for some phospholipids. However, in positive mode, non-native brominated adducts were found in LDL isolated using SBUC and evidence of potential bacterial contamination was discovered in samples prepared using IGUC, both of which have the capacity to affect in vitro experiments.