Radioactive and analytical determination of free and esterified cholesterol following micro thin-layer silicic acid chromatography

Vahouny, George V.; Borja, C.R.; Weersing, S.

doi:10.1016/0003-2697(63)90150-7

Cited by 39 publications

(7 citation statements)

References 4 publications

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“…Miscellaneous Procedures. Cholesterol and cholesteryl ester content of luteal plasma membranes and lipoproteins was determined according to Vahouny et al (1963). Phospholipid content was measured as previously described by us (Reaven & Azhar, 1981).…”

Section: Preparation Of Syntheticmentioning

confidence: 99%

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes

Shi¹,

Azhar²,

Reaven³

1992

Biochemistry

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Steroidogenic cells are able to utilize lipoprotein-derived cholesteryl esters for steroidogenesis without internalizing intact lipoproteins. In the current report, we provide evidence that an early step in this process may be the selective extraction of cholesteryl esters at the cell (plasma membrane) surface. We have used a highly purified plasma membrane preparation from rat luteinized ovaries for incubation with rat- and human-derived high density (HDL) and low density (LDL) lipoproteins. The lipoproteins were modified with residualizing [125I]apoprotein or [3H]cholesteryl ester markers. Following trypsin treatment to remove intact surface-bound apoprotein particles, the membranes were analyzed for transferred radioactive labels. The results show that all the lipoproteins tested could serve as cholesteryl ester donors. Although far more [3H]cholesteryl ester than [125I]apoprotein radioactivity was transferred to plasma membranes in each case, and varied with the ligand used, the total (net) mass of cholesteryl ester transferred was comparable with the different lipoproteins. These data were confirmed using direct chemical methodology. Transfer was found to be specific for cholesteryl esters or ethers and did not involve other lipoprotein core lipids tested. Endomembranes from the same tissue could not substitute for plasma membranes as the primary cholesteryl ester acceptor. These results provide evidence that a reconstituted lipoprotein-plasma membrane system can simulate the cholesteryl ester extraction process described in situ and suggest uses for this methodology in future experiments designed to understand the transfer process.

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Section: Preparation Of Syntheticmentioning

confidence: 99%

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes

Shi¹,

Azhar²,

Reaven³

1992

Biochemistry

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“…The digitonides were dissolved in methanol and counted in a proportional liquid scintillation counter using 10 ml of a scintillation mixture containing diphenyloxazole (4 gJl), POPOP/I (100 mg/l) and toluene. I n addition, difficultly-hydrolyzable C-MEC( 8) was determined directly chemically with Liebermann-Burchardt reagent (10) , after removal of digitonin-precipitable free and esterified cholesterol, and for radioactivity by the method of Vahouny et aZ (9).…”

Section: Methodsmentioning

confidence: 99%

“…The value of tritiated thymidine and autoradiography in the study of in v i m cell kinetics has been well established( 5-7). In vitro studies have also been used (8)(9)(10). The incubation of freshly drawn bone marrow with tritiated thymidine in vitro results in incorporation of the isotope into the nucleus of those cells that are synthesizing DNA.…”

Section: N Odartchenkoo and H Cottier11mentioning

confidence: 99%

Absolute Requirement for Free Sterol for Absorption by Rat Intestinal Mucosa.

Vahouny¹,

Treadwell²

1964

Experimental Biology and Medicine

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Several types of evidence indicate that the mechanism of transfer of cholesterol from the intestinal lumen into mucosal cells is specific for the free sterol. I t has been shown that there is extensive hydrolysis of fed cholesterol esters in the intestinal lumen, whereas essentially no esterification of dietary free cholesterol occurs intraluminarly ( 1 ) . Also, irrespective of whether free or esterified choles-ter01-4-C~~ is administered, only 2 5% of the labeled sterol in the intestinal mucosa is present in the esterified form, while in lymph, 80-90% exists as esterified cholesterol-4-C14 ( 2 ) . Thus, the apparent mechanism of absorption of either form is the same. Finally, the rate and extent of transfer of various cholesterol esters from lumen to thoracic duct lymph is directly related to the susceptibility of these esters to cholesterol esterase hydrolysis in vitro (3,4). Cholesterol trimethylacetate is not split enzymatically in vitro, and its administration in vivo does not produce a chemical increase in lymph cholesterol over a 24-hour period (3).These studies, however, have not provided direct evidence on the absorbability of intact, or unhydrolyzed, cholesterol esters into mucosal cells and lymph. The present study was undertaken to obtain such direct evidence. A s terically-hindered, disubsti tu ted fatty acid ester of cholesterol-4-C14 has been synthesized and studied for susceptibility to cholesterol esterase splitting in vitro, and absorbability in vivo using lymph fistula rats. Further, the ester was solubilized in phospholipid-bile salt micelles to provide an optimum medium for enzymatic hydrolysis in vivo (5). Materials and methods. Choles ter01-4-P~ a,a-methyl ethyl caproatet (C-MEC) wasCorp. ; a,a-methyl ethylcaproic acid, from Henley and Co., Inc., New York. prepared according to Swell and Treadwell ( 6 ) , and the reaction products were extracted in 20 volumes of 2 : 1 chloroform-methanol. After addition of water and separation of the phases, the chloroform extract was evaporated to dryness under nitrogen on a steam bath. The residue was reextracted into petroleum ether, the extract was dried over anhydrous sodium sulfate, and the ester was repeatedly purified by silicic acid column chromatography ( 7 ) . Purity was determined by methods described by Stern and Treadwell (8), and by quantitative micro-thin layer chromatography for free and esterified cholesterol-4-C14 (9). The final product had a melting point of 58"C, and contained 0.35% contamination of free cholesterol-4-C14.Phospholipid-bile salt micellar medium containing the labeled ester was prepared by homogenizing 6.8 mg C-MEC, 14 mg sodium taurocholate and 20 mg phosphatidyl choline per 5 ml isotonic saline. The mixture was sonicatedt for 30 minutes prior to use and had very slight opalescence (5).Adult male rats (225-250 g) of the Carworth strain were used and cannulation of the thoracic duct was performed as described earlier ( 10). T o avoid gastric alteration of the micellar dispersion, an infusion catheter was placed in the...

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“…This entire mixture was applied with 10,ug. each of standard cholecalciferol and cholecalciferol palmitate to micro-thinlayer plates (Vahouny, Borja & Weersing, 1963), which were developed with hexane-diethyl ether-acetic acid (20:4:1, by vol.). The micro-plates were stained with iodine vapour to locate free and esterified fractions and destained by heating at 500, and the silica gel was scraped quantitatively into two vials for measurement of the radioactivity in each area by liquid-scintillation spectrometry.…”

Section: Studie8 Of E8teriftcation In Vitromentioning

confidence: 99%

Enzyme studies on the esterification of vitamin D in rat tissues

Fraser

Kodíček

1968

Biochemical Journal

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1. The mechanism of vitamin D esterification in the rat was studied with liver, small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and [1-(3)H]cholecalciferol, [U-(14)C]ergocalciferol and [4-(14)C]cholesterol as substrates. 2. No esterification of vitamin D could be detected with liver preparations nor with homogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreatic juice esterified [1-(3)H]cholecalciferol with oleic acid, and specificity studies indicated that a cholesterol-esterifying enzyme was using vitamin D as substrate. 4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The specificity of the esterification reaction is discussed in relation to (a) the molecular structure of the substrates and (b) their availability, in a micellar solution, to the enzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin D in vivo during absorption from the small intestine and while it is transported in blood.

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Radioactive and analytical determination of free and esterified cholesterol following micro thin-layer silicic acid chromatography

Cited by 39 publications

References 4 publications

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes

Absolute Requirement for Free Sterol for Absorption by Rat Intestinal Mucosa.

Enzyme studies on the esterification of vitamin D in rat tissues

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