Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

327

292

This article is available online at http://www.jlr.org Circulating lipids, their biosynthesis, metabolism, and biological functions are intimately involved in many complex disease processes ( 1 ). Traditional clinical chemistry uses measurements of total cholesterol, triglycerides, and HDL as tools for determining health status and disease risk. The tests for these lipids are low cost, high throughput, and well established. The development of soft ionization techniques, particularly electrospray ionization has proven to be a watershed for lipidomics, allowing the detection and quantifi cation of individual molecular species. Recently, the Lipid Maps Consortium described a detailed analysis of the plasma lipidome, reporting on the concentration of nearly 600 lipids in pooled human plasma from healthy individuals ( 1, 2 ). This analysis highlighted the complexity of the plasma lipidome and the potential of plasma lipid profi ling for disease classifi cation, risk assessment, and to uncover changes in lipid metabolism associated with disease states. To date, plasma lipid profi ling has been used to identify lipidomic biomarkers associated with a variety of diseases and activities related to obesity ( 3 ), hypertension ( 4 ), smoking ( 5 ), cystic fi brosis ( 6 ), weight loss ( 7 ), and type 2 diabetes ( 8 ). These studies have, in general, have been conducted using relatively small cohorts (<100 participants) ( 3, 4, 6, 7 ) and/or limited coverage of the lipidome (<100 species) ( 4, 6, 8 ).Abstract We have performed plasma lipid profi ling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 l of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identifi ed statistically signifi cant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was signifi cantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk. -Weir, J. M., G.

Section: Associations Of Lipids With Anthropometric and Physiologicalmentioning

confidence: 99%

Plasma lipid profiling in a large population-based cohort

Weir

Wong

Barlow

et al. 2013

327

292

“…Cholesteryl esters (CEs), TAGs, and DAGs were extracted from weighed liver tissue (0.5-1 mg) suspended in 0.5 ml PBS that had been homogenized by sonication. Extractions of plasma (0.05 ml diluted to 0.1 ml with PBS), urine (1 ml), and tissue sonicates were carried out using 1 ml hexane:methyl t-butyl ether (1:1, v/v), essentially as previously described ( 31 ).…”

Section: Journal Of Lipid Research Volume 56 2015mentioning

confidence: 99%

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic

Gorden

Myers

Ivanova

et al. 2015

Self Cite

300

266

Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming one of the most common forms of liver disease in Abstract The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for defi nitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an "omics" approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profi ling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identifi cation of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of

“…Milk was collected from control, Spot14-null, and MMVTSpot14 overexpressing dams as previously described ( 46 ) and the neutral lipids were extracted, saponifi ed with NaOH to release individual fatty acids, and the fatty acid composition was quantifi ed by GC-MS according to previous methods ( 45,48,50 ). Loss of Spot14 resulted in signifi cantly decreased MCFA from the milk TAG at lactation day 10 ( Fig.…”

Section: Overexpression Of Spot14 Increases Mcfa Composition Of Milk Tagmentioning

confidence: 99%

“…HPLC grade reagents were purchased from Sigma-Aldrich, St. Louis, MO. Neutral lipid extraction was adapted from Hutchins et al ( 48,49 ), and pentafl uorobenzyl derivatization was according to Zarini et al ( 50 ). Mouse milk was thawed at 37°C for 10 min, vortexed, and 5 l of whole milk was transferred to 2 ml Eppendorf tubes containing 0.5 ml of 2:1 v/v methanol/ water; samples were acidifi ed with 40 l of 1M HCL, vortexed into solution, cleared by centrifugation at 10,000 g , and supernatant was transferred into borosilicate glass tubes.…”

Section: Milk Fat and Fatty Acid Composition Analysismentioning

confidence: 99%

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium

Rudolph

Wellberg

Lewis

et al. 2014

This article is available online at http://www.jlr.orgMammalian cells manufacture fatty acids de novo using a distinct pathway to synthesize fatty acids from acetyl and malonyl esters of CoA catalyzed by the dimer of FASN (EC 2.3.1.85) ( 1 ). FASN is absolutely essential for production of de novo fatty acids in mammals ( 2 ). The biological requirement for FASN is highlighted by the fact that FASN is detected in most normal human tissues and that homozygous deletion of FASN in the mouse results in embryonic lethality ( 3, 4 ). The importance of the de novo pathway is underscored by the complex biological functions of its fatty acid products, including biosynthesis of phospholipids, energy storage via esterifi cation into triglycerides (TAGs), use as energy substrates for ␤ -oxidation, providing both endocrine and nuclear hormone signaling ligands, and for critical posttranslational modifi cations of proteins ( 5-12 ). Perhaps the most demanding setting for FASN catalysis is during lactation, when extremely high quantities of de novo fatty acids are transmitted to the offspring as substrate for the growth of the young ( 13-15 ).Regulation of the principle enzymes of de novo fatty acid synthesis, ATP citrate lyase (ACLY) (EC 2.3.3.8), acetyl-CoA carboxylase (ACC) (EC 6.4.1.2), and FASN, has been shown to occur at the level of enzyme gene expression ( 16 ), translation/degradation ( 17-19 ), phosphorylation ( 20-22 ), assembly into multimeric complexes ( 23-25 ), and allosteric activation/inhibition ( 18,23,26,27 ). Recently, evidence for the control of de novo fatty acid synthesis by small effector proteins has emerged. Thyroid hormone responsive protein "Spot 14" (THRSP) (Spot14, S14) and only family Abstract Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefi ned during lactation. Spot14-null mice produce milk defi cient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When