An Untargeted Lipidomic Analysis Reveals Depletion of Several Phospholipid Classes in Patients with Familial Hypercholesterolemia on Treatment with Evolocumab

Rationale: Familial hypercholesterolemia (FH) is caused by mutations in genes involved in low-density lipoprotein cholesterol (LDL-C) metabolism, including those for pro-protein convertase subtilisin/kexin type 9 (PCSK-9). The effect of PCSK-9 inhibition on the plasma lipidome has been poorly explored. Objective: Using an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, the plasma lipidome of FH subjects before and at different time intervals during treatment with the PCSK-9 inhibitor Evolocumab was explored. Methods and Results: In 25 FH subjects, heterozygotes or compound heterozygotes for different LDL receptor mutations, untargeted lipidomic revealed significant reductions in 26 lipid classes belonging to phosphatidylcholine (PC), sphingomyelin (SM), ceramide (CER), cholesteryl ester (CE), triacylglycerol (TG) and phosphatidylinositol (PI). Lipid changes were graded between baseline and 4- and 12-week treatment. At 12-week treatment, five polyunsaturated diacyl PC, accounting for 38.6 to 49.2% of total PC at baseline; two ether/vinyl ether forms; seven SM; five CER and glucosyl/galactosyl-ceramide (HEX-CER) were reduced, as was the unsaturation index of HEX-CER and lactosyl—CER (LAC-CER). Although non quantitative modifications were observed in phosphatidylethanolamine (PE) during treatment with Evolocumab, shorter and more saturated fatty acyl chains were documented. Conclusions: Depletion of several phospholipid classes occurs in plasma of FH patients during treatment with the PCSK-9 inhibitor Evolocumab. The mechanism underlying these changes likely involves the de novo synthesis of SM and CER through the activation of the key enzyme sphingomyelin synthase by oxidized LDL and argues for a multifaceted system leading to vascular improvement in users of PCSK-9 inhibitors.

Insights into the lipidome and primary metabolome of the uterus from day 14 cyclic and pregnant sheep

In ruminants, conceptus elongation requires the endometrium and its secretions. The amino acid, carbohydrate, and protein composition of the uterine lumen during early pregnancy has been defined in sheep; however, a comprehensive understanding of metabolomic changes in the uterine lumen is lacking, particularly with respect to lipids. Here, the lipidome and primary metabolome of the uterine lumen, endometrium, and/or conceptus was determined on day 14 of the estrous cycle and pregnancy. Lipid droplets and select triglycerides were depleted in the endometrium of pregnant ewes. In contrast, select ceramides, diglycerides, and non-esterified fatty acids as well as several phospholipid classes (phosphatidylcholine, phosphatidylinositol, phosphatidylglycerols, and diacylglycerols) were elevated in the uterine lumen of pregnant ewes. Lipidomic analysis of the conceptus revealed that triglycerides are particularly abundant within the conceptus. Primary metabolite analyses found elevated amino acids, carbohydrates, and energy substrates, among others, in the uterine lumen of pregnant ewes. Collectively, this study supports the hypothesis that lipids are important components of the uterine lumen that govern conceptus elongation and growth during early pregnancy.

Characterization of the Lipidome and Biophysical Properties of Membranes from High Five Insect Cells Expressing Mouse P-Glycoprotein

The lipid composition of biomembranes influences the properties of the lipid bilayer and that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High Five™ insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 ± 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.

Characterization of the Lipidome and Biophysical Properties of Membranes from High Five Insect Cells Expressing Mouse P-Glycoprotein

The lipid composition of biomembranes influence the properties of the lipid bilayer as well as that of the proteins. In this study, the lipidome and the lipid/protein ratio of membranes from High FiveTM insect cells overexpressing mouse P-glycoprotein was characterized. This provides a better understanding of the lipid environment in which P-glycoprotein is embedded, and thus of its functional and structural properties. The relative abundance of the distinct phospholipid classes and their acyl chain composition was characterized. A mass ratio of 0.57 +/- 0.11 phospholipids to protein was obtained. Phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. Membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositols and phosphatidylglycerols), and contain small amounts of sphingomyelins, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated, with significant amounts of saturated chains. The characterization of the phospholipids by HPLC-MS allowed identification of the combination of acyl chains, with palmitoyl-oleoyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. A mixture of POPE:POPC:POPS in the ratio 45:35:20 is proposed for the preparation of simple representative model membranes. The adequacy of the model membranes was further evaluated by characterizing their surface potential and fluidity.

Separation, identification and cardiovascular activities of phospholipid classes from the head of Penaeus vannamei by lipidomics and zebrafish models

Five phospholipid classes of Penaeus vannamei head were separated, analyzed and quantified. They had different cardiovascular activities evaluated in zebrafish models, which may provide a research basis for pharmaceutical use of marine phospholipids.

Protocols for Enzymatic Fluorometric Assays to Quantify Phospholipid Classes

Phospholipids, consisting of a hydrophilic head group and two hydrophobic acyl chains, are essential for the structures of cell membranes, plasma lipoproteins, biliary mixed micelles, pulmonary surfactants, and extracellular vesicles. Beyond their structural roles, phospholipids have important roles in numerous biological processes. Thus, abnormalities in the metabolism and transport of phospholipids are involved in many diseases, including dyslipidemia, atherosclerosis, cholestasis, drug-induced liver injury, neurological diseases, autoimmune diseases, respiratory diseases, myopathies, and cancers. To further clarify the physiological, pathological, and molecular mechanisms and to identify disease biomarkers, we have recently developed enzymatic fluorometric assays for quantifying all major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol + cardiolipin, and sphingomyelin. These assays are specific, sensitive, simple, and high-throughput, and will be applicable to cells, intracellular organelles, tissues, fluids, lipoproteins, and extracellular vesicles. In this review, we present the detailed protocols for the enzymatic fluorometric measurements of phospholipid classes in cultured cells.

Modifications of Membrane Phospholipids in Response to Extended Aging from Pork Loins

ObjectivesIt is well established that fresh meat shelf-life deteriorates during aging process. We hypothesize that part of the shelf-life reduction is due to membrane phospholipid deterioration through phospholipase activity and/or phospholipid oxidation during aging. Therefore, the objective of this study was to characterize the modifications/deterioration of phospholipid classes/species in pork loins from 3 different aging periods.Materials and MethodsLoins from 20 carcasses were collected at a commercial harvest facility in the Midwest 1 d postmortem from carcasses of Duroc sired crossbred pigs. Four chops from each carcass containing only the longissimus muscle were vacuum packaged and aged at 4°C for 1, 8, and 21 d. A sensitive approach based on electrospray ionization tandem mass spectrometry was used to comprehensively analyze phospholipid composition using the lipid extract from each sample at each aging period (n = 60). Unsaturation index (UI; measurement of the number of double bonds) for each phospholipid species class was also calculated to quantify fatty acyl chain unsaturation for each sample in each aging period.ResultsTotal phospholipid quantity in pork loins was not different between 1d and 8d aged chops but decreased significantly from 8d to 21d of aging (806.6 vs. 297.5 nmol phospholipid/mg lipid; P < 0.01). On the other hand, the mol% data (distribution of each phospholipid species in relative % of total phospholipid) revealed that phosphatidylinositol (PI) and phosphatidylserine (PS) increased in mol% from 1d to 21d of aging in pork loins (P < 0.01). This increase was mainly due to the increase of PI 38:4 (primarily 18:0/20:4) and PS 36:2 (primarily 18:0/18:2) between 1d and 21d samples (P < 0.01). The results showed that phospholipid degradation products like lysophosphatidylcholine (LPC) mol% rose quickly after short term aging (8d) but remained constant through the rest of the 21d aging period (P < 0.01). Conversely, lysophosphatidylethanolamine (LPE) was unaltered between 1d and 8d of aging but decreased between 8d and 21d aged pork loins (P < 0.01). The mol% of phosphatidic acid (PA) also increased between 1d and 21d aged pork loins (P < 0.05). Extended aging did not alter the mol% of total phosphatidylcholine (PC), ether-linked PC (ePC), sphingomyelin (SM), phosphatidylethanolamine (PE) or ether-linked PE (ePE; P > 0.05). Surprisingly, UI revealed the exact opposite trend as the mol% data. The UI of PI and PS decreased (P < 0.01) from 1d to 21 d of aging in pork loins due to the disappearance of many minor PI and PS species with very long chain fatty acids and multiple double bonds such as PI 42:10 and PS 44:10. There was also a slight increase of PC UI after 8 d of aging in pork loins (P < 0.01). The UI for LPC, ePC, SM, LPE, PE, ePE and total phospholipid were not altered in any of the aging periods (P > 0.05).ConclusionThese results confirmed our hypothesis that phospholipids undergo extensive degradation during aging. The data also indicated that the majority of phospholipids in pork loins may maintain integrity over short period aging (1–8d). Among the phospholipid classes, PI and PS were slightly more resistant to deterioration compared with the others due to their ability to modify fatty acyl chain saturation. Additional investigations are necessary to define the role of phospholipid modifications in fresh pork shelf-life and flavor.