Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids

Silversand, Christer; Haux, Carl

doi:10.1016/s0378-4347(97)00385-x

Cited by 111 publications

(88 citation statements)

References 23 publications

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“…The phosphatidylethanolamine species were synthesized from corresponding phosphatidylcholines using phospholipase D-mediated transphosphatidylation (31). The lipids were purified using normal-phase high pressure liquid chromatography on a diol-modified silica column (32). The purity of phospholipids was confirmed by TLC and mass spectrometry, and their concentrations were determined based on phosphate content (33).…”

Section: Methodsmentioning

confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9 -10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA 2 s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA 2 s are key players.Type A phospholipases (PLAs) 3 are ubiquitous enzymes involved in many biological phenomena, such as signal transduction (1) and phospholipid homeostasis (2), including acyl chain remodeling (3) and degradation (4). However, the identities of the specific proteins involved in these phenomena have often not been established. PLAs are also clinically relevant because they have been implicated in many common diseases or disorders, including atherosclerosis, allergy, Alzheimer disease, and cancer (5).Several PLAs display significant specificity toward the phospholipid acyl chain(s) in vitro (6, 7). However, despite numerous studies, the factors underlying such specificity have remained largely elusive. In principle, two factors contribute to selective hydrolysis of phospholipids by PLA: 1) accommodation of the acyl chains in the protein lipid binding site and 2) the ease of efflux of the phospholipid substrate from the bilayer (8, 9). The understanding of acyl chain accommodation has been greatly hampered by the lack of crystal structures of PLAs complexed with a physiological substrate. Crystal structures have been obtained for some PLAs with a bound noncleavable shortchain substrate analogue (10 -13), but because these analogues have truncated acyl chains, they only provide very limited information on the factors underlying acyl chain specificity. Furthermore, the information could also be biased, since studies with other lipid-binding proteins have shown that the mode of accommodation of an alkyl...

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Section: Methodsmentioning

confidence: 99%

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Haimi¹,

Hermansson²,

Batchu³

et al. 2010

Journal of Biological Chemistry

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“…The PE and PS species with a deuterium-labeled head group were synthesized from corresponding phosphatidylcholine species and D 4 -ethanolamine or D 3 -serine, respectively, using phospholipase D-mediated transphosphatidylation as described previously for unlabeled lipids (27) except that the reaction volume was reduced 5-fold. The products were purified by normal phase HPLC (28), and their purity was confirmed by mass spectrometry. PE and PS labeled both to the fatty acid and head group were prepared by acylating sn1-oleyl-or sn1-palmitoyl-lysoPC by [ 13 C 18 ]oleate (29) followed by conversion of the sn1-oleoyl-and sn1-palmitoyl- [2-13 C 18 ]oleyl-PC to corresponding PE or PS species as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Quantification was carried out using three internal standards for both PE and PS (36). For identification of the acyl chain sn-positions of the endogenous species, as well as the remodeling products derived from the exogenous species, PE and PS classes were isolated by normal phase HPLC (28), and the individual species were then subjected to collisionally activated dissociation (CAD) and product ion analysis. Albeit some previous and ESI-MS studies have indicated that it is possible to identify the sn-positions of the acyl chain of phospholipids by collisional fragmentation and product ion scanning (37)(38)(39), controversial data exist (39,40).…”

Section: Methodsmentioning

confidence: 99%

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

Kainu

Hermansson

Somerharju

2008

Journal of Biological Chemistry

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Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation, and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely because of limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class-specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis.

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“…Further development was achieved with a polyvinyl alcohol-bonded phase (PVA), that was used for the separation of sphingolipids, glycoglycerolipids, phospholipids and the more polar lipid constituents of marine particulate matter [9,10]. Other authors presented methods for the separation of neutral and polar lipid classes using diol-NP columns [11,12]. They demonstrated excellent separations either for neutral lipids or polar lipids.…”

Section: Introductionmentioning

confidence: 99%

Improved separation and quantification of neutral and polar lipid classes by HPLC–ELSD using a monolithic silica phase: Application to exceptional marine lipids

Graeve

Janssen

2009

Journal of Chromatography B

124

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a b s t r a c tAn improved HPLC method is presented, which allows separation and quantification of a broad range of lipid classes of marine zooplankton with special regard to neutral lipids. Marine zooplankton species often produce high amounts of exceptional lipids, especially at high latitudes, in order to cope with the harsh environmental conditions and strong seasonality in food supply. Major neutral lipid classes are wax esters, triacylglycerols, diacylglycerol ethers, free fatty alcohols and sterols. Neutral and polar lipids were separated and identified on a monolithic silica column (Chromolith ® Performance-Si) using high performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). The method resolves a broad spectrum of lipids, varying in polarity from squalene to lysophosphatidylcholine in a single run. The total run time was 35 min including column re-equilibration. The calibration was made at levels of 0.1-60 g lipid/injection, but a 10-15-fold greater amount can be injected if single lipid classes need to be separated, e.g. for further determination of individual fatty acids. The method was applied to representative Arctic zooplankton species (copepods, pteropods, euphausiids and ctenophores) that are known to biosynthesize in particular neutral lipids like diacylglycerol ethers and free fatty alcohols.

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Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids

Cited by 111 publications

References 23 publications

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

Improved separation and quantification of neutral and polar lipid classes by HPLC–ELSD using a monolithic silica phase: Application to exceptional marine lipids

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