Lipidyzer platform

Serum lipids were analyzed using the direct infusion-tandem mass spectrometry (DI-MS/MS) Lipidyzer platform (Sciex, MA, USA) that identifies and quantifies ~1,100 biological lipids covering 13 lipid classes (i.e. free fatty acids, ceramides, diacylglycerols, triacylglycerols). The Lipidyzer platform methodology has been described in detail elsewhere [99 ], but briefly, lipids were extracted from 100 μL of serum using a modified Bligh-Dyer method. Over 50 stable isotope labeled internal standards spanning all 13 lipid classes were added to each sample prior to extraction for accurate quantitation. Extracts were reconstituted in dichloromethane/methanol (1:1) and analyzed using DI-MS/MS with DMS separation. A Shimadzu LC system was used for automated infusion of each serum extract and for pumping running and rinse solutions through the lines. Serum extracts were infused into a 5500 QTRAP MS/MS with SelexION DMS technology (Sciex) and lipid species were targeted and quantitated using optimized MS/MS transitions. Data were generated using the Lipidomics Workflow Manager software (Sciex). Results provided the concentration (μM) and fatty acid composition (mol%) of total lipid classes and individual lipid species.

Lipids were extracted from plasma using the Bligh-Dyer method as previously described^{56 (link)}. The extracted dry lipids were resuspended in 250 µl running buffer (10 mM ammonium acetate and 50:50 methanol:dichloromethane). Lipids were analyzed on a Sciex Lipidyzer Platform with a standardized workflow for the simultaneous analysis of 1,153 lipids representing 13 lipid class. Samples were loaded by direct infusion from a Shimadzu LC-30AD LC system equipped with a SIL-30AC auto sampler. Lipid concentrations were determined by the Lipidyzer software using the ratio of the endogenous lipid to internal standard. Data are reported for each individual lipid species as an aggregated value for lipid classes and as the relative composition compared to all other measured lipid classes.

Samples were analyzed on the Sciex Lipidyzer Platform (Framingham, MA, USA) and quantified with normalization to serum volume. This platform has been described elsewhere [58 ]; briefly, it is a direct infusion-tandem mass spectrometry system utilizing differential mobility. Analysis is carried out over two infusions, with each acquiring roughly half of the lipid targets within the assay. As many as 1100 targeted lipid species can be identified using the Lipidyzer targeted multiple-reaction monitoring (MRM) list, but only 622 were quantified in at least 33% of cases or 33% of controls. The cut point of 33% was chosen to avoid correlational artifact weighted by lipids detected in very few samples. This allowed us to examine nearly 57% of the 1100 targeted lipid species in a sample that was less likely to have missing data. A total of 352 lipids had no missing data in both cases or controls (i.e. were quantified for all 198 samples tested), of which 251 were TAGs. Concentrations were reported as nmoles/mL. Lipid class totals were calculated in each sample by summing the concentration of all individual lipid species within each class and presented in units of nanomoles/mL.

We used Fisher Optima^TM grade reagents for all sample preparation and LC mobile phases (Fisher Scientific, Hanover Park, IL, USA). Chemical standards for urine metabolomics included debrisoquine sulfate and 4-nitrobenzoic acid for internal standards, and carnitine, trigonelline hydrochloride, xanthurenic acid, Nε,Nε,Nε-trimethyllysine hydrochloride, and spermine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hexosamine-valine-isoleucine-OH (Hex-V-I) was synthesized by Expert Synthesis Solutions (London, ON, Canada) and structure confirmation has been previously published [8 (link)]. Chemical standards for serum lipidomics included EquiSPLASH^® LIPIDOMIX, (d₇) 15:0/18:1 phosphatidic acid, (d₇) 18:1 cholesteryl ester (CE) (Avanti Polar Lipids Inc., Alabaster, AL, USA), the free fatty acid (FFA), dihydroceramide (DCER), hexosylceramide (HexCer), and lactosylceramides (LCER) internal standard kits for the Lipidyzer™ Platform (Sciex, Framingham, MA, USA), and the NIST plasma Standard Reference Material (SRM) 1950.