Measurement of different lipid species using mass spectrometry

High performance liquid chromatography (HPLC)-grade water, ethanol, and methanol were purchased from Fisher Scientific (Schwerte, Germany). HPLC-grade pyridine, phenyl isothiocyanate (PITC) and ammonium acetate were acquired from Merck (Darmstadt, Germany). The following standards from Avanti Polar Lipids were used for normalization: 06:0 PC (DHPC), 19:0 Lyso PC, 08:0 PE, 06:0 SM (d18:1/6:0), and Splash II Lipidomix Mass Spec Internal Standard. The carnitine standards octanoyl-L-carnitine d3 and palmitoyl-L-carnitine d3 were purchased from Supelco Analytical.

Mouse brain and liver samples were mechanically homogenized in water via Minilys (PEQLAB, Erlangen, Germany) for 60 s on maximum intensity. Protein was measured using bicinchoninic acid assay according to Smith et al.⁸⁵ and homogenates were adjusted to a protein amount of 10 mg/ml in water.

The used solid/liquid lipid extraction method is described in detail in Grimm et al.²⁴. Briefly, a 96 well filter plate (0.45 μm; Merck) was fixed on a 96-deep well plate (Fisher Scientific) and circles of whatman blotting paper with a diameter of 6 mm were placed into the wells of the filter plate. On these Whatman papers a standard mixture was added, followed by 10 μl of each prepared sample (described above). After drying the samples under a nitrogen flow (1–2 bar) for 45 min, 20 μl of 5% PITC (v/v) diluted in ethanol / water / pyridine (1:1:1, v/v/v) were added to the wells and incubated for 20 min at room temperature. Samples were again dried for 45 min under nitrogen, before lipids were extracted by the use of 300 μl 4.93 mM ammonium acetate in methanol and shaking the plate for 30 min at 450 rpm on a plate shaker (IKA, Staufen, Germany). Liquid samples were transferred into the 96-deep well plate by centrifugation for 2 min at 500×g. Afterwards, the samples were diluted with 600 μl 5 mM ammonium acetate in methanol/water (97:3, v/v), the plate was covered with a silicone mat and shaken for further 2 min at 450 rpm and room temperature before mass spectrometry analysis. An average extraction efficiency of > 80.7% (intra-day variance of 3.9%) and a linearity of R² > 0.96 for this lipid extraction method were determined for these experimental conditions (see supplemental Figure ^S5D).

For measurement of different species of diacyl-phosphatidylcholines (PC aa), phosphatidylcholine-plasmalogens (PC ae), lyso-phosphatidylcholines (Lyso PC), acyl- and acetyl-carnitines, sphingomyelins (SM) and triglycerides (TAG) a 4000-quadrupole linear-ion trap (QTrap) equipped with a Turbo Spray ion source (AB Sciex, Darmstadt, Germany) was used. Detection of different lipid species was carried out in triplicates using the Analyst 1.4.2 software (AB Sciex, Darmstadt, Germany) with help of an autosampler of the Agilent HPLC 1200. Lipid analysis was performed in positive mode using the following parameters: measurement period = 3 min, scan type = multiple reaction monitoring (MRM), curtain gas = 20.0 psi, collision gas = medium, ion spray voltage = 5500.0 V, temperature = 200.0 °C, ion source gas 1 = 40 psi, ion source gas 2 = 50 psi, interface heater = on, entrance potential = 10 V, collision cell exit potential = 15 V. The used Q1 and Q3 masses, declustering potentials (DP) and collision energies (CE) for each metabolite were derived, among others, from^24,86 and are listed in supplementary table ¹. The constantly measured intra- and inter-day variance was in average 6.5% (see supplemental Figure ^S5A). Potential matrix effects were evaluated by calculating the ratio between the deuterated lipid standards in presence of lipid extracts from acitretin-treated and control mice. The change in the ratio was maximum 3.2% and in average 1.4% as shown in supplemental Figure ^S5B.