Improved procedure for the extraction of lipids from human erythrocytes

Rose, Herbert G.; Oklander, Morris

doi:10.1016/s0022-2275(20)39314-7

Cited by 974 publications

(72 citation statements)

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“…Lipids were removed from red cell membranes by an isopropanol-chloroform extraction procedure. 4 The precipitate which remained (mainly the protein portion of the membrane) was about 50% soluble in 2-chloroethanol. After removal of the solvent from the extracted membrane lipids, these were dissolved in 2-chloroethanol for ORD study.…”

mentioning

confidence: 99%

Protein Conformation in Cell Membrane Preparations as Studied by Optical Rotatory Dispersion and Circular Dichroism

Lenard

Singer

1966

Proc. Natl. Acad. Sci. U.S.A.

301

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To elucidate the basic structure of biological membranes, experimental evidence concerning the detailed molecular organization of the proteins of membranes is required. This evidence has been almost totally lacking or ambiguous up to the present time. The Cotton effects due to the peptide bond ultraviolet absorption bands, as studied by ORD and CD, have recently proved extremely valuable in characterizing the molecular conformations of polypeptides and soluble proteins. In this paper, we report the results of ORD and CD studies in the ultraviolet on membrane preparations from human red blood cells and from B. subtilis in several solvent media.Materials and Methods.-Membrane preparations: Red blood cell membranes were prepared by the method of Dodge et al.1 from fresh human blood. Heme analyses' of several preparations showed uniformly that no more than 0.5% of the total protein present was hemoglobin. Membrane preparations from B. subtilis, strain 168, were prepared essentially by the procedure of Salton and Ehtisham-Ud-Din.' A low-speed centrifugation step was inserted to remove bacterial debris before the 39,000 X g centrifugation that brought down the membranes.3 Microscopic examination of both of these membrane preparations showed them to consist almost entirely of whole membranes. Solutions of each membrane preparation in 2-chloroethanol were prepared at 40 from lyophilized portions of the preparations.Lipids were removed from red cell membranes by an isopropanol-chloroform extraction procedure.4 The precipitate which remained (mainly the protein portion of the membrane) was about 50% soluble in 2-chloroethanol. After removal of the solvent from the extracted membrane lipids, these were dissolved in 2-chloroethanol for ORD study. Lipid concentrations were determined by the organic phosphorus assay of Bartlett.'Reagents: Reagent grade 2-chloroethanol from several commercial sources was purified by distillation under reduced pressure. Sufficient concentrated hydrochloric acid was added to the purified 2-chloroethanol to give a pH of 2.69-2.71 when 1 vol of 2-chloroethanol was diluted with 9 vol of water." ORD and CD measurements and calculations: ORD and CD measurements were carried out at room temperature with a Durrum-Jasco UV-5 instrument.7 To calculate [m'], the reduced mean residue rotation, and 0, the mean molar ellipticity, the protein concentration is required. This was determined from the OD280 of 2-chloroethanol solutions prepared from measured amounts of the original membrane suspension. The OD280 of the 2-chloroethanol solution was related to the protein content by total acid hydrolysis and amino acid analysis on a modified Beckman 120 analyzer.8 The protein concentration was then calculated by summing all the amino acid residues and assuming a mean residue weight of 114. The refractive indices of the aqueous media were assumed to be identical to pure water.9 For 2-chloroethanol, the refractive index measurements of Foss and Schellman'0 were extrapolated into the wavelength region of interest.Resul...

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mentioning

confidence: 99%

Protein Conformation in Cell Membrane Preparations as Studied by Optical Rotatory Dispersion and Circular Dichroism

Lenard

Singer

1966

Proc. Natl. Acad. Sci. U.S.A.

301

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“…During our method development, Rose & Oklander (Rose and Oklander, 1965) and Folch (Folch et al 1957) methods for lipid extraction were evaluated. The percentage of lipids extracted using single extractions was 69-75% by Rose and Oklander's method and 63-72% by Folch's method.…”

Section: Resultsmentioning

confidence: 99%

“…Lipids were extracted from 200 μl red blood cells in glass tubes with Teflon-lined screw caps by adding isopropanol with continuous vortexing. Subsequently, chloroform was added in ratio 11:7, by volume (Rose and Oklander, 1965) with 50 mg/l of butylated hydroxy toluene (BHT) as antioxidant (to prevent autoxidation of PUFA). One hundred microliters of C17:0 was added to every sample as internal standard (0.1 mg/ml).…”

Section: Extraction Of Lipidsmentioning

confidence: 99%

Standardization and validation of assay of selected omega-3 and omega-6 fatty acids from phospholipid fraction of red cell membrane using gas chromatography with flame ionization detector

et al. 2021

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Background Docosahexaenoic acid (DHA) is an important structural component of human brain and retina. Evidence exists linking nutritional status of pregnant mothers and cognitive functions of their born infants. The DHANI (Maternal DHA Supplementation and Offspring Neurodevelopment in India) trial was implemented to evaluate the effect of maternal supplementation with DHA during pregnancy and for 6 months following delivery on motor and mental development of infants at 1 and 12 months. We describe here the standardization and validation of an assay for measurement of selected omega-3 and omega-6 fatty acids from the phospholipid fraction of red blood cells to assess their status in mothers at baseline, delivery and 6 months post-delivery and for infants in cord blood and at 1 and 12 months of age. The validated method has been used for the analysis of samples for DHANI. Methods Lipids were extracted from a pool of red blood cells, separated using thin layer chromatography. The phospholipid fraction was esterified, and fatty acids were separated by gas chromatography using a flame ionization detector. Result The method accuracy for DHA was between 97 - 98% and between 91 - 95% for arachidonic acid (AA) at three different concentrations. The intra-assay and inter-assay coefficient of variation for the fatty acids ranged from 1.19 to 5.7% and 0.78 to 13.0% respectively. Intraclass correlation (ICC), as a measure of reproducibility, ranged between 0.689 and 0.996. A good linearity was observed for all the fatty acids between concentrations of 0.2–4 μg/ml. Conclusion The standardized and validated method is suitable for implementation in large epidemiological studies for evaluation of fatty acids and in nutritional trials for assessment of fatty acid content of various lipid classes.

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“…Three microliters of butylated hydroxytoluene (88 mg/1 ml absolute alcohol), an antioxidant, were added to 500-,u 1 aliquots of ghosts prepared from erythrocytes of control and poisoned sheep and stored at -20°C . Lipid extraction from erythrocyte membranes was performed as described by Rose and Oklander [12]. Separation of the various phospholipids in the lipid extract was carried out by TLC on silica gel H glass plates (silica 60 H, 0.5 mm thickness, Merck Laboratory) with a chloroform/methanol/glacial acetic acid/ water solvent system (50: 25: 8 : 4; v/v).…”

Section: Methodsmentioning

confidence: 99%

Lipid Peroxidation in Erythrocyte Membranes of Sheep with Chronic Copper Poisoning.

Sansinanea¹,

Cerone²,

Najle³

et al. 1994

J. Clin. Biochem. Nutr.

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Corriedale ewes were exposed to ration and a pasture containing a high level of copper to study the relationship between peroxidation of lipid membrane and copper-induced hemolysis. We measured phospholipids, cholesterol, and protein from red blood cell (RBC) ghosts, and lipid peroxidation. A decrease in total phospholipids in the erythrocyte membranes and an increase in lipid peroxide levels of erythrocytes was observed in blood samples from animals that ingested large amount of copper. The cholesterol concentration was not altered in RBC. Additionally, depressed phospholipid/protein and phospholipid/ cholesterol ratios were found. These data suggest that elevated copper levels have a deleterious effect on membrane integrity through the peroxidation of lipids. It is reasonable to postulate that these alterations were a possible cause of hemolysis.

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Improved procedure for the extraction of lipids from human erythrocytes

Cited by 974 publications

References 9 publications

Protein Conformation in Cell Membrane Preparations as Studied by Optical Rotatory Dispersion and Circular Dichroism

Protein Conformation in Cell Membrane Preparations as Studied by Optical Rotatory Dispersion and Circular Dichroism

Standardization and validation of assay of selected omega-3 and omega-6 fatty acids from phospholipid fraction of red cell membrane using gas chromatography with flame ionization detector

Lipid Peroxidation in Erythrocyte Membranes of Sheep with Chronic Copper Poisoning.

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