Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots

Rouser, George; Fleischer, Sidney; Yamamoto, Akira

doi:10.1007/bf02531316

Cited by 3,224 publications

(1,561 citation statements)

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“…Small PEG and non-PEG liposomes were sized to a diameter between 90 nm and 100 nm, while the diameter of large PEG liposomes was set between 450 nm and 500 nm. Phospholipid content was determined with a phosphate assay (16) in the organic phase after extraction of liposomal preparations with chloroform. The aqueous phase after extraction was used for determining the PLP content.…”

Section: Methodsmentioning

confidence: 99%

Complete remission of experimental arthritis by joint targeting of glucocorticoids with long‐circulating liposomes

Metselaar¹,

Wauben²,

Wagenaar-Hilbers³

et al. 2003

Arthritis & Rheumatism

281

224

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Objective. To increase the therapeutic activity of glucocorticoids in experimental arthritis by encapsulation in long-circulating polyethylene glycol liposomes, which have shown the ability to preferentially accumulate in inflamed joints after intravenous administration.Methods. Rats with adjuvant-induced arthritis (AIA) were treated intravenously with liposomal and free prednisolone phosphate (PLP) a few days after the first signs of disease. The effect on paw inflammation scores during the weeks after treatment was evaluated. Liposome biodistribution and joint localization were investigated by labeling the preparation with radioactive 111 In-oxine. By studying PLP encapsulated in other types of liposomes, which show a distinctive tissue distribution pattern and reduced accumulation in inflamed joints, the importance of targeted delivery to inflamed joints for achieving an increased therapeutic effect was illustrated.Results. Liposomal PLP proved to be highly effective in the rat AIA model. A single injection of 10 mg/kg resulted in complete remission of the inflammatory response for almost a week. In contrast, the same dose of unencapsulated PLP did not reduce inflammation, and only a slight effect was observed after repeated daily injections. Evidence was found that preferential glucocorticoid delivery to the inflamed joint was the key factor explaining the observed strong therapeutic benefit obtained with the liposomal preparation, while other possible mechanisms, such as splenic accumulation or prolonged release of prednisolone in the circulation, were excluded.Conclusion. Targeted delivery using longcirculating liposomes is a promising, novel means to successfully intervene in arthritis with glucocorticoid therapy.

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Section: Methodsmentioning

confidence: 99%

Complete remission of experimental arthritis by joint targeting of glucocorticoids with long‐circulating liposomes

Metselaar¹,

Wauben²,

Wagenaar-Hilbers³

et al. 2003

Arthritis & Rheumatism

281

224

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“…Phosphoenhancement of either membrane bilayer fluidity [26] or phoslipids were extracted from membranes according to Bligh and Dyer pholipid substrate accessibility for the enzyme. An increase in [20] and quantified by inorganic phosphorus measurements, as described by Rouser et al [21]. The number of -SH groups in rat liver rotational motion of enzymatic protein and in phospholipid microsomes was determined spectrophotometrically at 412 nm, by transbilayer movement cannot be excluded, either.…”

Section: R Jasihska Et Ai/febs Letters 386 (1996) 33-38mentioning

confidence: 99%

Nonenzymatically evoked and cytochrome P450‐dependent lipid peroxidation inhibits synthesis of phosphatidylethanolamine via the ethanolamine base exchange reaction in rat liver microsomes

et al. 1996

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In the present study the relationship between lipid sole pathway for PS formation. Sundler et al. [3] reported that peroxidation, changes in the redox state of membrane and in isolated rat hepatocytes, at physiological concentrations of phosphatidylethanolamine (PE) synthesis via base exchange ethanolamine (25 ~tM), 8-9% of the total PE synthesis could reaction in rat liver microsomes was investigated. It was found be attributed to Ca2+-dependent incorporation of ethanolthat PE synthesis is enhanced in the presence of antioxidants, amine via the BE reaction. The reaction does not result in a butylated hydroxytoluene (BHT), or unsaturated free fatty acids, net increase of the total PE content, but is rather responsible Prooxidants, tert-butyl hydroxyperoxide (BHP), ferrous ions for significant remodelling of preexisting membrane phosphocombined with ascorbate or NADPH (via cytochrome P450-lipids. The most abundant molecular species of PS and PE are dependent proteins), increased the amount of lipid peroxidation known to contain long-chain polyunsaturated fatty acids: araproducts in the membrane, and in consequence inhibited the chidonic (20:4), docosatetraenoic (22:4)and docosahexaenoic reaction. The effect of BHP was fully reversed by reduced (22:6) in the sn-2 position of their glycerol moiety [4]. glutathione and dithiothreitol (DTT), whereas the effect of other compounds could be reversed only by BHT. In contrast, a Furthermore, Ellingson and Seenaiah [5] have documented reversal of the inhibitory effect of cadmium ions on base that stearoyl-polyunsaturated molecular species of PE and exchange activity was observed in the presence of DTT, but PC are preferentially converted to PS by the phospholipid not BHT. Therefore, both the -SH/-S-S-ratio in the membrane, BE reaction in rat liver microsomes. affected by BlIP and cadmium ions, and the lipid hydroxyper-PE, kept in a bilayer configuration by interactions with oxides (rather than aldehydes), generated by ferrous ions and other membrane phospholipid molecules, is able to induce ascorbate or NADPH, are equally responsible for the inactivalocal nonbilayer structures by creating hexagonal phases [6]. tion of the ethanolamine base exchange enzyme in rat liverThis property of PE may be responsible for regulation of the microsomes. This may suggest that the synthesis of PE via the activity of many membranous enzymes, for example cytobase exchange reaction may be considered an element of the chrome P450 [7]. The latter class of enzymes participates in superfine cellular machinery involved in the repair of damage to unsaturated fatty acid chains of phospholipids caused by reactive hydroxylation processes of different hydrophobic molecules, oxygen species under oxidative stress, like fatty and bile acids, phospholipids, steroid hormones and/or xenobiotics [8]. Moreover, the PE hexagonal phase

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“…Lipids were extracted from the erythrocytes according to the procedure of Rose and Oklander [24] and separated by two-dimensional thin-layer chromatography according to the method of Broekhuyse [25]. The specific radioactivity of the erythrocyte PC was determined using established procedures for determination of phosphorus [29], and radioactivity (see above). This specific activity can be directly correlated with the extent of PC replacement when the specific activity of the donor PC at the start of the incubation is known and when the radioactively labeled PC is representative for the bulk of donor PC.…”

Section: Preparation Of the Transfer Proteinmentioning

confidence: 99%

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

Kuypers

Roelofsen

Kamp

et al. 1984

Biochimica et Biophysica Acta (BBA) - Biomembranes

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(1) Using the phosphatidylcholine specific transfer protein from bovine liver, native phosphatidylcholine from intact human erythrocytes was replaced by a variety of different phosphatidylcholine species without altering the original phospholipid and cholesterol content. (2) The replacement of native phospharidylcholine by the disaturated species, 1,2-dipalmitoyl-and 1,2-distearoylphosphatidyicholine, proceeded at a low rate and extensive replacement could only be achieved by repeatedly adding fresh donor vesicles. The replacement by disaturated molecules was accompanied by a gradual increase in osmotic fragility of the cells, finally resulting in hemolysis when 40% of the native PC had been replaced. Up to this lyric concentration, the replacement did not affect the permeability of the membrane for potassium ions. (3) Essentially, all of the PC in the outer monolayer of the membrane could be replaced by 1-palmitoyl-2-oleoyl-and 1-palmitoyl-2-1ino-leoylphosphatidylcholine. These replacements did not alter the osmotic fragility of the cells, nor the K + permeability of the membrane. (4) Increasing the total degree of unsaturation of the phosphatidyicholine species modified the properties of the membrane considerably. Replacement by 1,2-dilinoleoylphosphatidylcholine resulted in a progressive increase in osmotic fragility and hemolysis started to occur after 30% of the native PC had been replaced by this species. K ÷ permeability was found to be slightly increased in this case. Cells became leaky for K ÷ upon the introduction of 1-palmitoyl-2-arachidonoylphosphatidyicholine in the membrane. The increased permeability was also reflected by an apparent increase in the resistance of the cells against osmotic shock. (5) The conclusions to be drawn are that (i) 1-palmitoyl-2-oleoyi-and l-paimitoyi-2-1inoleoylphosphatidyicholine are species which fit most optimally into the erythrocyte membrane; (ii) loss of membrane stability results from an increase in the degree of saturation of phosphatidyicholine (unsaturation index > 0.5) and (iii) the permeability is enhanced by increasing the content of highly unsaturated species (unsaturation index > 1.0).

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Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots

Cited by 3,224 publications

References 1 publication

Complete remission of experimental arthritis by joint targeting of glucocorticoids with long‐circulating liposomes

Complete remission of experimental arthritis by joint targeting of glucocorticoids with long‐circulating liposomes

Nonenzymatically evoked and cytochrome P450‐dependent lipid peroxidation inhibits synthesis of phosphatidylethanolamine via the ethanolamine base exchange reaction in rat liver microsomes

The membrane of intact human erythrocytes tolerates only limited changes in the fatty acid composition of its phosphatidylcholine

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