Improved separation of phospholipids in thin layer chromatography

Touchstone, Joseph C.; Chen, John C.; Beaver, Kathleen M.

doi:10.1007/bf02534120

Cited by 474 publications

(180 citation statements)

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“…Two dimensional TLC requires about 320 nmol of total phospholipid to visualize the individual phospholipids (23). Nevertheless, it should be noted that the use of special mobile phases containing triethylamine in one-dimensional high performance TLC/densitometry allows complete separation of the six major phospholipids present in amniotic fluid, with similar detection limits to those reported here, but requires a prior lipid extraction step (11). On the other hand, our enzymatic methods can be applied directly to aqueous samples of membranes, body fluids and other biological materials without previous lipid extraction.…”

Section: Resultssupporting

confidence: 51%

“…Currently, numerous methods for the estimation of phospholipids in body fluids and other biological materials are available. The most frequently used involves the separation of different classes of phospholipids by TLC and their quantification by densitometry or by measuring the phosphorus content of each phospholipid (6)(7)(8)(9)(10)(11). Accurate phospholipid values can also be obtained with the aid of HPLC (12,13).…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Determination of Phosphatidylcholine, Sphingomyelin and Phosphatidylglycerol in Lipid Dispersions, Blood Cell Membranes and Rat Pulmonary Surfactant

Encinar

Ludeña²,

Sánchez‐Yagüe³

et al. 1996

CCLM

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Summary: A new micromethod for the determination of sphingomyelin in samples suspended in aqueous solutions, and modified micromethods for determining phosphatidylcholine and phosphatidylglycerol were used to determine phosphatidylcholine and sphingomyelin (detection limits of 1.8 μπιοΙ/1), and phosphatidylglycerol (detection limit of 2.3 μιηοΐ/l) in lipid dispersions, membranes from sheep erythrocytes and platelets, and pulmonary surfactants from rats of different ages and rats maintained under normobaric hyperoxia for 2 days prior to their sacrifice. The procedures are easy to perform, accurate, require less sample than conventional methods and can also be applied directly to aqueous samples. Phospholipase C and sphingomyelinase were used to release phosphorylcholine from phosphatidylcholine and sphingomyelin, respectively. The choline released from phosphorylcholine by alkaline phosphatase is reconverted to phosphorylcholine by ATP and choline kinase. In the phophatidylglycerol determination, phospholipase D was used to release glycerol and phosphatidate. The glycerol formed was converted to glycerolphosphate using ATP and glycerol kinase. In all cases, the ADP thus formed was determined by following the enzymatic conversion of NADH to NAD at 340 nm in an coupled pyruvate kinase/lactate dehydrogenase system. Significant variations in the phospholipid composition of rat pulmonary surfactant were found during development; in particular there was an increase in the phosphatidylglycerol content of adult rats as compared with younger rats. Hyperoxia produced changes in the phosphatidylglycerol content of surfactant from adult rats, but not from 2-day old rats.

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Section: Resultssupporting

confidence: 51%

Section: Introductionmentioning

confidence: 99%

Enzymatic Determination of Phosphatidylcholine, Sphingomyelin and Phosphatidylglycerol in Lipid Dispersions, Blood Cell Membranes and Rat Pulmonary Surfactant

Encinar

Ludeña²,

Sánchez‐Yagüe³

et al. 1996

CCLM

View full text Add to dashboard Cite

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“…The composition of CLSE by weight was 93% phospholipid, 4.5% cholesterol, 1.5% protein (a mixture of SP-B and SP-C), and 1% other. The phospholipid class distribution was ϳ85% PC, 5% PG, 4% PI (including phosphatidylserine), 3% PE, 1% Sph, and 2% other (51). ELISA testing using a monoclonal antibody to bovine SP-B (rabbit derived) has reported a level of 0.9% SP-B by weight relative to phospholipid in CLSE material similar to that used here (39).…”

Section: Methodsmentioning

confidence: 68%

“…Cholesterol was reagent grade from Sigma (St. Louis, MO). DPPC was Ͼ99% pure as supplied and gave a single well-defined spot on thin-layer chromatography with solvent system C of Touchstone et al (51). Egg PC, egg PG, egg PI, egg PE, and Sph gave broader single spots on thinlayer analysis, consistent with their content of multiple fatty chain derivatives.…”

Section: Methodsmentioning

confidence: 72%

Content-dependent activity of lung surfactant protein B in mixtures with lipids

Wang

Baatz

Holm

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

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The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B ≈ SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels ≤0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.

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“…TLC plates were developed in a solvent system containing chloroform : methanol : triethylamine : 1-propanol : 0.25% (w/v) KCl (30 : 9 : 18 : 25 : 6, by vol.) for approximately 90 min (Touchstone et al 1980). TLC plates were air-dried for 5 min and visualized with 0.1% ANSA.…”

Section: Lipid Analysismentioning

confidence: 99%

Dietary low linolenic acid compared with docosahexaenoic acid alter synaptic plasma membrane phospholipid fatty acid composition and sodium–potassium ATPase kinetics in developing rats

Bowen

Clandinin

2002

Journal of Neurochemistry

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The objective of this study was to investigate if maternal dietary 20:4n-6 arachidonic acid (AA) and 22:6n-3 compared with adequate or low levels of 18:3n-3 linolenic acid (LNA) increases synaptic plasma membrane (SPM) cholesterol and phospholipid content, phospholipid 20:4n-6 and 22:6n-3 content, and Na,K-ATPase kinetics in rat pups at two and five weeks of age. At parturition, Sprague-Dawley rats were fed semi-purified diets containing either AA + docosahexaenoic acid (DHA), adequate LNA (control; 18:2n-6 : 18:3n-3 ratio of 7.1 : 1) or low LNA (18:2n-6 : 18:3n-39 ratio of 835 : 1). During the first two weeks of life, the rat pups received only their dams' milk. After weaning, pups received the same diet as their respective dams to five weeks of age. No significant difference was observed among rat pups fed the diet treatments for SPM cholesterol or total and individual phospholipid content at two and five weeks of age. Fatty acid analysis revealed that maternal dietary AA + DHA, compared with feeding the dams the control diet or the low LNA diet, increased 20:4n-6 in phosphatidylserine and 22:6n-3 content of SPM phospholipids. Rats fed dietary AA + DHA or the control diet exhibited a significantly increased V max for SPM Na,K-ATPase. Diet treatment did not alter the K m (affinity) of SPM Na,K-ATPase in rat pups at two and five weeks of age. It is concluded that dietary AA + DHA does not alter SPM cholesterol and phospholipid content but increases the 22:6n-3 content of SPM phospholipids modulating activity of Na,K-ATPase.

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Improved separation of phospholipids in thin layer chromatography

Abstract: The mobile phases described permit separation of the six major phospholipids of amniotic fluid in one dimension with either conventional or high performance thin layer chromatography. An example of this separation with an extract of amniotic fluid is given.

Cited by 474 publications

References 3 publications

Enzymatic Determination of Phosphatidylcholine, Sphingomyelin and Phosphatidylglycerol in Lipid Dispersions, Blood Cell Membranes and Rat Pulmonary Surfactant

Enzymatic Determination of Phosphatidylcholine, Sphingomyelin and Phosphatidylglycerol in Lipid Dispersions, Blood Cell Membranes and Rat Pulmonary Surfactant

Content-dependent activity of lung surfactant protein B in mixtures with lipids

Dietary low linolenic acid compared with docosahexaenoic acid alter synaptic plasma membrane phospholipid fatty acid composition and sodium–potassium ATPase kinetics in developing rats

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