Seufert Amy L, Hickman James W, Choi Jaewoo, Napier Brooke A
Department of Biology and Center for Life in Extreme Environments, Portland State University, Portland, OR, USA.
Linus Pauling Institute, Oregon State University, Corvallis, OR, USA.
Bio Protoc. 2023 Sep 20;13(18):e4819. doi: 10.21769/BioProtoc.4819.
Dietary saturated fatty acids (SFAs) are upregulated in the blood circulation following digestion. A variety of circulating lipid species have been implicated in metabolic and inflammatory diseases; however, due to the extreme variability in serum or plasma lipid concentrations found in human studies, established reference ranges are still lacking, in addition to lipid specificity and diagnostic biomarkers. Mass spectrometry is widely used for identification of lipid species in the plasma, and there are many differences in sample extraction methods within the literature. We used ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS) to compare relative peak abundance of specific lipid species within the following lipid classes: free fatty acids (FFAs), triglycerides (TAGs), phosphatidylcholines (PCs), and sphingolipids (SGs), in the plasma of mice fed a standard chow (SC; low in SFAs) or ketogenic diet (KD; high in SFAs) for two weeks. In this protocol, we used Principal Component Analysis (PCA) and R to visualize how individual mice clustered together according to their diet, and we found that KD-fed mice displayed unique blood profiles for many lipid species identified within each lipid class compared to SC-fed mice. We conclude that two weeks of KD feeding is sufficient to significantly alter circulating lipids, with PCs being the most altered lipid class, followed by SGs, TAGs, and FFAs, including palmitic acid (PA) and PA-saturated lipids. This protocol is needed to advance knowledge on the impact that SFA-enriched diets have on concentrations of specific lipids in the blood that are known to be associated with metabolic and inflammatory diseases. Key features • Analysis of relative plasma lipid concentrations from mice on different diets using R. • Lipidomics data collected via ultra-high performance liquid chromatography (UPLC) coupled to a high-resolution hybrid triple quadrupole-time-of-flight (QToF) mass spectrometry (MS). • Allows for a comprehensive comparison of diet-dependent plasma lipid profiles, including a variety of specific lipid species within several different lipid classes. • Accumulation of certain free fatty acids, phosphatidylcholines, triglycerides, and sphingolipids are associated with metabolic and inflammatory diseases, and plasma concentrations may be clinically useful.
膳食饱和脂肪酸（SFA）在消化后会在血液循环中上调。多种循环脂质种类与代谢和炎症性疾病有关；然而，由于人体研究中血清或血浆脂质浓度存在极大变异性，除了脂质特异性和诊断生物标志物外，目前仍缺乏既定的参考范围。质谱法广泛用于鉴定血浆中的脂质种类，文献中的样品提取方法存在许多差异。我们使用超高效液相色谱（UPLC）与高分辨率混合三重四极杆-飞行时间（QToF）质谱（MS）联用，比较了喂食标准饲料（SC；SFA含量低）或生酮饮食（KD；SFA含量高）两周的小鼠血浆中以下脂质类别内特定脂质种类的相对峰丰度：游离脂肪酸（FFA）、甘油三酯（TAG）、磷脂酰胆碱（PC）和鞘脂（SG）。在本方案中，我们使用主成分分析（PCA）和R来可视化个体小鼠如何根据其饮食聚类在一起，并且我们发现与喂食SC的小鼠相比，喂食KD的小鼠在每个脂质类别中鉴定出的许多脂质种类显示出独特的血液特征。我们得出结论，两周的KD喂养足以显著改变循环脂质，其中PC是变化最大的脂质类别，其次是SG、TAG和FFA，包括棕榈酸（PA）和PA饱和脂质。该方案对于推进关于富含SFA的饮食对已知与代谢和炎症性疾病相关的血液中特定脂质浓度的影响的认识是必要的。关键特征 • 使用R分析不同饮食的小鼠的相对血浆脂质浓度。 • 通过超高效液相色谱（UPLC）与高分辨率混合三重四极杆-飞行时间（QToF）质谱（MS）联用收集脂质组学数据。 • 允许对饮食依赖性血浆脂质谱进行全面比较，包括几种不同脂质类别中的多种特定脂质种类。 • 某些游离脂肪酸、磷脂酰胆碱、甘油三酯和鞘脂的积累与代谢和炎症性疾病有关，血浆浓度可能具有临床用途。
