Webb Richard J, Lodge John K, Scott Sophie S, Davies Ian G
School of Health and Sport Sciences, Liverpool Hope University, Liverpool L16 9JD, UK.
School of Human Sciences, London Metropolitan University, London N7 8DB, UK.
Metabolites. 2025 Jan 22;15(2):68. doi: 10.3390/metabo15020068.
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).
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.
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 ( < 0.001).
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.
背景/目的：基于盐的密度梯度超速离心法（SBUC）常用于分离脂蛋白以便后续分析。然而，盐的添加可能会破坏其分子组成。因此，本研究的目的是评估与经过验证的非盐方法——碘克沙醇梯度超速离心法（IGUC）相比，SBUC对低密度脂蛋白（LDL）颗粒分子组成的影响。
在使用SBUC和IGUC方法分离LDL颗粒之前，对全人血浆进行各种脂质参数分析。然后对每个组分进行过滤以获得低分子量化合物。使用非靶向液相色谱 - 质谱法（LC - MS）以正离子和负离子模式测定两种方法所得组分中的LDL分子含量。
分别使用正离子和负离子模式推测鉴定出总共1041个和401个特征峰。使用SBUC和IGUC制备的LDL分子组成存在差异；在正离子模式电离下，偏最小二乘判别分析（PLS - DA）模型显示出合理的拟合度和判别能力（R2 = 0.63，Q2 = 0.58，准确率0.88），且置换检验具有显著性（<0.001）。
研究结果揭示了使用这两种方法制备的LDL小分子组成存在明显差异，IGUC表现出更大的变异性。在负离子模式下，两种方法都检测到了磷脂、长链鞘脂和神经酰胺，但IGUC对某些磷脂显示出更高的倍数差异。然而，在正离子模式下，在使用SBUC分离的LDL中发现了非天然溴化加合物，并且在使用IGUC制备的样品中发现了潜在细菌污染的证据，这两者都有可能影响体外实验。
