High-Resolution Separation and Quantification of Neutral Lipid and Phospholipid Species in Mammalian Cells and Sera by Multi-One-Dimensional Thin-Layer Chromatography

White, Thayer; Bursten, Stuart L.; Federighi, David; Lewis, Robert A.; Nudelman, Edward

doi:10.1006/abio.1997.2545

Cited by 166 publications

(110 citation statements)

References 31 publications

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“…The cells were incubated in fresh media for 6 -48 h, then washed five times with phosphate-buffered saline, before addition of isopropyl alcohol (1 ml). After 16 h at 4°C the extracted lipids were dissolved in chloroform (150 l) and aliquots, together with standards, analyzed on TLC plates as described (37,38). Areas corresponding to specific lipid species were recovered after exposure of the plates to x-ray film and radioactivity determined using scintillation counting.…”

Section: Animals-abcg1mentioning

confidence: 99%

Deletion of the Transmembrane Transporter ABCG1 Results in Progressive Pulmonary Lipidosis

Baldán

Tarr

Vales³

et al. 2006

Journal of Biological Chemistry

106

127

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We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1 ؊/؊ mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3-and 8-month-old Abcg1 ؊/؊ mice. These data suggest that the lungs of young Abcg1 ؊/؊ mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1 ؊/؊ , but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1 ؊/؊ mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.

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Section: Animals-abcg1mentioning

confidence: 99%

Deletion of the Transmembrane Transporter ABCG1 Results in Progressive Pulmonary Lipidosis

Baldán

Tarr

Vales³

et al. 2006

Journal of Biological Chemistry

106

127

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“…To determine relative contents of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, and sphingomyelins, lipids were extracted into chloroform, loaded as 0.5-cm bands on TLC plates, and separated using methyl acetate/n-propanol/chloroform/methanol/ 0.25% aqueous potassium chloride (25:25:25:10:9 by volume) as the solvent system (35). Plates were dried and sprayed with a 0.05% solution in acetone/water (80:20 by volume) of the lipid-binding fluorescent dye primulin (Sigma) (36). An image of the silica plate was generated by laser-excited fluorescence detection scanning using a STORM 840 PhosphorImager (Molecular Dynamics), and ImageQuant™ software was used to integrate intensities of individual bands.…”

Section: Lipid Analysesmentioning

confidence: 99%

Phosphatidylcholine Transfer Protein Promotes Apolipoprotein A-I-mediated Lipid Efflux in Chinese Hamster Ovary Cells

Baez

Barbour

Cohen

2002

Journal of Biological Chemistry

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Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein of unknown function that catalyzes intermembrane transfer of phosphatidylcholines in vitro.Using stably transfected CHO cells, we explored the influence of PC-TP on apolipoprotein A-I-and high density lipoprotein 3 (HDL 3 )-mediated lipid efflux. In proportion to its cellular level of expression, PC-TP accelerated apolipoprotein A-I-mediated phospholipid and cholesterol efflux as pre-␤-HDL particles. PC-TP increased rates of efflux of both lipids by >2-fold but did not affect mRNA levels or the activity of ATP-binding cassette A1, a plasma membrane protein that regulates apolipoprotein A-I-mediated lipid efflux. Overexpression of PC-TP was associated with only slight increases in HDL 3 -mediated phospholipid efflux and no changes in cholesterol efflux. In scavenger receptor BI-overexpressing cells, PC-TP expression minimally influenced apolipoprotein A-I-or HDL 3 -mediated lipid efflux. PC-TP did not affect cellular phospholipid compositions, phosphatidylcholine contents, or phosphatidylcholine synthetic rates. These findings suggest that a physiological function of PC-TP is to replenish the plasma membrane with phosphatidylcholines that are removed during pre-␤-HDL particle formation due to the activity of ATP-binding cassette A1.

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“…A solvent mixture of 80:20:1 hexane/diethyl ether/acetic acid was used for lipid separation. After drying, the plate was sprayed with 0.05% primulin dye dissolved in 80:20 acetone/water [32]. The plate was illuminated with UV light and photographed using an Alpha Imager 2200 (Alpha Innotech Corp., San Leandro, CA).…”

Section: Thin Layer Chromatographymentioning

confidence: 99%

Microplate assay for quantitation of neutral lipids in extracts from microalgae

Higgins¹,

Thornton-Dunwoody²,

Labavitch³

et al. 2014

Analytical Biochemistry

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Lipid quantitation is widespread in the algae literature but popular methods including gravimetry, GC-MS, and Nile red cell staining suffer drawbacks including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that utilizes Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75-40 µg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement to TAG levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely-used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

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High-Resolution Separation and Quantification of Neutral Lipid and Phospholipid Species in Mammalian Cells and Sera by Multi-One-Dimensional Thin-Layer Chromatography

Cited by 166 publications

References 31 publications

Deletion of the Transmembrane Transporter ABCG1 Results in Progressive Pulmonary Lipidosis

Deletion of the Transmembrane Transporter ABCG1 Results in Progressive Pulmonary Lipidosis

Phosphatidylcholine Transfer Protein Promotes Apolipoprotein A-I-mediated Lipid Efflux in Chinese Hamster Ovary Cells

Microplate assay for quantitation of neutral lipids in extracts from microalgae

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