Lipid metabolism of leukocytes in the unstimulated and activated states

Abstract

Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting-edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently, differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the Lipidyzer™ platform to obtain a comprehensive overview of leukocyte-related lipid metabolism in the resting and activated states. First, we tested the linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+), we shed light on leukocyte-specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at the resting state, PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ cells and PMNs, whereas CD14+ cells did not show significant changes. By exploring the fatty acid content of the significantly upregulated lipid classes, we mainly found increased concentrations of very long and polyunsaturated fatty acids. Our results indicate the usefulness of the Lipidyzer™ platform for studying cellular lipid metabolism. Its application allowed us to explore the lipidome of leukocytes.

Lipidomic Phenotyping Reveals Extensive Lipid Remodeling during Adipogenesis in Human Adipocytes

“…In order to study the process of adipogenesis of human SGBS cells we decided to use the novel targeted Lipidyzer™ technology. Although originally developed and validated for the fast and automated analysis of human plasma samples [ 20 ], recently published studies also showed good performance of the Lipidyzer™ assay with other matrices than plasma [ 21 , 22 , 23 , 24 , 25 ]. With the aim to obtain a meaningful “fit for purpose method”, we investigated the analytical performance regarding linearity and repeatability for the SGBS cells.…”

“…Prior to the adipogenesis characterization, we analyzed the method performance in terms of linearity, repeatability, and background signals to be able to apply this novel methodology for accurate lipid analysis to SGBS cell culture samples. We used representative samples for undifferentiated and differentiated SGBS cells and validated the Lipidyzer™ method according to recently published studies that already showed good performance of the Lipidyzer™ method [ 21 , 22 , 23 , 24 , 25 ].…”

Obesity-Related Changes in Human Plasma Lipidome Determined by the Lipidyzer Platform

“…Lipids were extracted from human plasma samples using a modified Bligh-Dyer method [ 16 , 18 , 19 , 20 ]. Briefly, plasma samples of 100 µL were mixed with 900 µL of HPLC grade water, 2000 µL of methanol, and 900 µL of dichloromethane in glass centrifuge tubes and vortexed for 5 s. To prepare a quality control sample, 100 µL of quality control plasma (QC) was mixed with 900 µL of HPLC grade water, 2000 µL of methanol, and 900 µL of dichloromethane in another glass centrifuge tube and vortexed for 5 s. To make a spiked sample, 50 µL of quality control spike standard (QC spike) was mixed with 100 µL of QC, 900 µL of HPLC grade water, 2000 µL of methanol, and 900 µL of dichloromethane in a separate glass centrifuge tube and vortexed for 5 s. To compensate for background FA contamination, a blank sample was also prepared by mixing 1000 µL of HPLC grade water, 2000 µL of methanol, and 900 µL of dichloromethane in a glass centrifuge tube and vortexed for 5 s. Then, 100 µL of ISTD mixture was added to each sample (plasma samples, quality control sample, spiked sample, blank sample), vortexed for 5 s, and incubated at room temperature for 30 min.…”

Reproducibility of Targeted Lipidome Analyses (Lipidyzer) in Plasma and Erythrocytes over a 6-Week Period

“…Next, FFA, TAG, DAG, CER, dihydroceramide (DCER), lactosylceramide (LCER), hexosylceramide (HCER), and CE lipids were analyzed applying method #2. Further technical detail including a list of all monitored transitions and detailed experimental setting can be found elsewhere [ 24 , 25 , 26 ]. In the erythrocyte samples, lipid concentrations were corrected for the erythrocyte protein pellet content, which was quantified using a Micro BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).…”