Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids

Bodennec, Jacques; Pelled, Dori; Futerman, Anthony H.

doi:10.1194/jlr.d200026-jlr200

Cited by 22 publications

(21 citation statements)

References 60 publications

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“…During a manual procedure, reextraction of the water phase is accurate extraction of these lipid classes might be achieved by using the initial one-phase extract for further analysis without performing the two-phase extraction step or by adding extraction steps on top of the BUME method. It may well be possible to use a solvent mixture based on a high proportion of butanol for additional automated extractions of very polar lipids in the aqueous phase from the original BUME method, similar to what has previously been described ( 27 ). However, the development of a method for the extraction of very polar lipids lies outside the scope of this report and needs to be investigated further.…”

Section: Discussionmentioning

confidence: 99%

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Löfgren

Ståhlman

Forsberg

et al. 2012

Journal of Lipid Research

273

209

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This article is available online at http://www.jlr.org implicated in several diverse diseases, such as type 2 diabetes, Alzheimer disease, ( 2 ) and cancer ( 3 ). Lipid analysis has been an important area of research for several decades, and due to technological advances, the fi eld has experienced a renaissance in the last decade. In a modern laboratory, a comprehensive lipid characterization can be performed that generates quantitative data of several hundreds of molecular lipids from several different lipid classes. This kind of analysis, often called lipidomics, is based on HPLC and mass spectrometry (MS) instrumentation. The analysis is performed unattended in the 96-well format and is fully automated.An important component for successful analysis is the quality of the lipid extract. It is important that the lipid extract that is injected on the HPLC or infused into the mass spectrometer is pure; therefore, it is important that interfering substances and particles are removed. Inability in removing these substances might result in a high chemical background, which will have an effect on both the sensitivity and selectivity of the analysis. In contrast to fully automated and high-throughput analysis, lipid extraction is still often performed manually, involving exhaustive and time-consuming pipetting steps and hazardous solvents such as chloroform. Thus, a fully automated, chloroformfree method that can be used with standard 96-well robots would signifi cantly improve sample throughput, as well as reduce the negative impact on health and environment. The aim of this study was to develop that method.Abstract Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profi les of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroformbased reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by twophase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, highthroughput BUME method can replace chloroform-based methods, saving both human and environment...

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Section: Discussionmentioning

confidence: 99%

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Löfgren

Ståhlman

Forsberg

et al. 2012

Journal of Lipid Research

273

209

View full text Add to dashboard Cite

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“…During a 60-min incubation at 60°C, the liberated CH 3 ONH 2 gas derivatized the ketone or aldehyde groups present on the oxidized phospholipids. Following derivatization, the dried lipids were suspended in 200 l of CHCl 3 and applied to a hexane-conditioned aminopropyl Sep-Pak (NH 2 -SPE) column to separate lipids by class (25,26). The neutral lipids were eluted with 4 ml of CHCl 3 , isopropyl alcohol, 2:1 (v/v).…”

Section: Methodsmentioning

confidence: 99%

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Frasch

Berry

Fernandez-Boyanapalli

et al. 2008

Journal of Biological Chemistry

102

141

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Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lysoPS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91 phox؊/؊ ). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91 phox؊/؊ mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.Neutrophils are often robustly recruited early in inflammation. Within hours of their activation in tissues, they are removed by phagocytes, an event required for resolution of inflammation and the return to normalcy of tissue function. It is known that neutrophils undergoing apoptosis drive the production of anti-inflammatory mediators such as transforming growth factor-␤ that actively suppress production of inflammatory cytokines, chemokines, eicosanoids, and nitric oxide (1, 2). Indeed, enhanced induction of neutrophil apoptosis in vivo is potently anti-inflammatory (3, 4). However, if recognition and clearance fail, activated and dying neutrophils ultimately disintegrate releasing injurious intracellular constituents (e.g. serine proteases) (5). Failure of timely cell clearance is associated with both autoimmunity and enhanced inflammation (6, 7).Phosphatidylserine (PS) 2 exposed in the plasma membrane outer leaflet of apoptotic cells has long been known as a key ligand important for their recognition and removal. Interaction with various PS receptors, including the recently identified TIM4 (8, 9), BAI1 (10), and stabilin 2 (11) or PS-recognizing bridge molecule-receptor combinations (e.g. MFG-E8 and ␣ v integrins or Gas6 and Mer (12)), have been demonstrated. In many, bu...

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“…Lipid Analysis-Lipid analysis was done as described (3), except extractions included back-extraction with water-saturated butanol to avoid loss of more polar oligogalactolipid species, as described previously (57). The thin layer chromatography (TLC) liquid phases used were chloroform, methanol, 0.45% (w/v) NaCl in water (60:35:8, v/v/v), or chloroform/ methanol/acetate/water (85:20:10:4, v/v/v/v).…”

Section: Alignment and Selection Of Crystal Structure Templates Formentioning

confidence: 99%

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

Roston

Wang

Kuhn

et al. 2014

Journal of Biological Chemistry

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Background: SENSITIVE TO FREEZING 2 (SFR2) is classified as a glycosyl hydrolase, and by using glycosyltransferase activity, it modifies membrane lipids to promote freeze tolerance. Results: Although the active site of SFR2 is identical to hydrolases, adjacent loop regions contribute to its transferase activity. Conclusion: Transferase activity evolved by modifications external to the core catalytic site. Significance: Defined structure-function relationships will inform engineering of transferases and freeze tolerance.

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Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids

Cited by 22 publications

References 60 publications

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase

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