Computational Analysis of Plasma Lipidomics from Mice Fed Standard Chow and Ketogenic Diet

Abstract

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.

Keywords: Circulating lipids; Free fatty acids; Ketogenic diet; Lipidomics; Mass spectrometry; Phosphatidylcholines; Sphingolipids; Triglycerides.

Figure 1.. Isobaric structure of triglyceride as shown by UPLC-QToF MS/MS: TAG (16:0/18:1/18:2) or TAG (16:0/18:2/18:1).

Separation and identification of isobaric lipids with identical molecular formulas is limited due to structural differences that do not show chromatographic separation. (A) Extract ion chromatogram of TAG (16:0/18:1/18:1) or TAG (16:0/18:2/18:1). (B) ToF-MS isotopic pattern of m/z 874.7858. (C) ToF-MS/MS of m/z 874.7858, which shows ammonium adduct in positive ion mode.

Figure 2.. Lipidomics data processing

Figure 3.. Lipid [PC (16:0/18:2)] identification using PeakView software, which explores and interprets qualitative data.

(A) Extract ion chromatogram (XIC) of PC (16:0/18:2). (B) XIC manager displays in the table including found mass, mass error, found retention time, formula, adduct, and exact mass. (C) TOF-MS isotopic pattern of m/z 758.5694, which shows PC (16:2/18:0). (D) TOF-MS/MS of m/z 758.5694 as protonated adduct. The m/z 184 represents a unique fragment ion (protonated phosphocholine) corresponding to phosphatidylcholine.

Figure 4.. Chromatographic peak area integration using MultiQuant software.

(A) Analyte pane includes identified PCs list. (B) Result pane includes each sample name and peak area counts with retention time. (C) Chromatogram review in each sample. The peak can be automatically or manually integrated. AUC values are highlighted in yellow.

Figure 5.. Lipidomics data analysis (see General note 4).

(A) PCA plot of phosphatidylcholine composition identified via LC-QToF MS/MS between age-matched (6–8 weeks) BALB/c female mice. Dots represent individual mice with colors corresponding to a standard chow (SC) (grey) or ketogenic diet (KD) (orange) with the mean of each diet group surrounded by a 95% confidence ellipse. (B) Biplot labeled with the top five phosphatidylcholines contributing to sample separation in 2D space. (C) Heatmap analysis of phosphatidylcholine composition between individual mice.