Enzyme Electrode with Collagen-Immobilized Cholesterol Oxidase for the Microdetermination of Free Cholesterol

To test the validity of filipin cytochemistry for localization of cholesterol in testicular cells, we compared the results obtained by this technique with those obtained by a two-step enzymatic method involving cholesterol esterase and cholesterol oxidase. In all the animals models tested (guinea pig, mink, and mallard duck) the disappearance of subsurface filaments along Sertoli cell junctional membranes was accompanied by a significant increase in the number of filipin-cholesterol complexes/microns 2 in these membranes. Enzyme histochemistry allowed localization of free cholesterol in the limiting membrane of multivesicular bodies, in membranes within lysosomes, in mitochondrial membranes, and in junctional membranes, with or without subsurface filaments. The method also permitted selective visualization of cholesterol esters in lipid droplets. We conclude that filipin mapping of cholesterol induces false-negative cytochemical results. The enzymatic method is superior to filipin because it allows localization of free cholesterol in junctional membranes and of cholesterol esters in lipid droplets. This compartmentalization of the compounds may represent the basis of a system that helps to maintain constant free cholesterol levels in the testis.

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Enzymatic Determination of Total Serum Cholesterol

Clinical Chemistry ◽

10.1093/clinchem/20.4.470 ◽

1974 ◽

Vol 20 (4) ◽

pp. 470-475 ◽

Cited By ~ 4700

Author(s):

Charles C Allain ◽

Lucy S Poon ◽

Cicely S G Chan ◽

W Richmond ◽

Paul C Fu

Keyword(s):

Serum Cholesterol ◽

Free Cholesterol ◽

Cholesterol Oxidase ◽

Total Serum ◽

Total Serum Cholesterol ◽

Enzymatic Method ◽

Simultaneous Production ◽

Enzymatic Determination ◽

Production Of Hydrogen

Abstract An enzymatic method is described for determination of total serum cholesterol by use of a single aqueous reagent. The method requires no prior treatment of sample and the calibration curve is linear to 600 mg/dl. Cholesterol esters are hydrolyzed to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol produced is oxidized by cholesterol oxidase to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a chromogen with maximum absorption at 500 nm. The method is reproducible, and the results correlate well with those obtained by automated Liebermann—Burchard procedures (AA-2 and SMA 12/60) and the method of Abell et al. The present method affords better specificity than those previously reported and has excellent precision.

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Automated enzymic measurement of total cholesterol in serum.

Clinical Chemistry ◽

10.1093/clinchem/24.1.108 ◽

1978 ◽

Vol 24 (1) ◽

pp. 108-114 ◽

Cited By ~ 36

Author(s):

G S Rautela ◽

R J Liedtke

Keyword(s):

Hydrogen Peroxide ◽

Total Cholesterol ◽

Response Surface ◽

Computer Analysis ◽

Free Cholesterol ◽

Cholesterol Oxidase ◽

Cholesterol Esters ◽

Direct Proportion ◽

Analytical Test ◽

Response Surface Designs

Abstract We describe a completely automated enzymic system for measuring total cholesterol in serum. All reagents are contained in an analytical test pack and the test is performed on Du Pont's Automatic Clinical Analyzer (aca), which mixes the sample (20 microliter) and reagents and performs the necessary absorbance measurements and calculations. In the procedure, cholesterol oxidase oxidizes free cholesterol. The oxidation step produces cholest-4-en-3-one and hydrochloride peroxidesterase hydrolyzes cholesterol esters and cholesterol in direct proportion to the amount of cholesterol present. N,N-Diethylaniline hydrochloride and 4-aminoantipyrine react with the hydrogen peroxide to produce a quinoneimine dye (lambda max = 553 nm). Interacting reagents have been optimized simultaneously (coöptimization) utilizing response surface designs coupled with computer analysis of the data. Reagent efficiency is high and analytical performance reliable.

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Abstract 392: The Role of Scavenger Receptor-BI’s Transmembrane Domains in Cholesterol Transport: A "Locked Dimer" Strategy

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.392 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Sarah Proudfoot ◽

Alexandra Chadwick ◽

Emma Allen ◽

Daisy Sahoo

Keyword(s):

Disulfide Bonds ◽

Free Cholesterol ◽

Cholesterol Oxidase ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Immunoblot Analysis ◽

Surface Expression ◽

Transmembrane Domains ◽

Cell Surface Expression ◽

Cholesterol Transport

Efficient reverse cholesterol transport requires interactions between high density lipoprotein (HDL) and its receptor, scavenger receptor-BI (SR-BI). SR-BI is an 82 kDa protein with a large extracellular domain anchored by two transmembrane domains (TMDs). Our lab recently solved the NMR structure of SR-BI’s C-terminal TMD (C-TMD), a region that mediates SR-BI dimerization. Further, FRET studies suggest HDL-induced movement between neighboring SR-BI monomers, which led to our hypothesis that flexibility between SR-BI TMDs facilitates cholesterol transport. Using structure-guided mutagenesis, we introduced cysteine residues into the C-TMD of full-length SR-BI to create “locked dimers” of the receptor. Total lysate and cell surface expression of WT-, A444C-, L451C-, or G453C-SR-BI were verified in transiently-transfected COS-7 cells by immunoblot analysis and flow cytometry, respectively. Based on the predicted orientation of sulfhydryl side chains relative to the putative dimerization motif, we used immunoblot analysis following electrophoresis under reducing/non-reducing conditions to confirm that A444C- and L451C-SR-BI, but not G453C-SR-BI, formed disulfide bonds. Compared to WT-SR-BI, the locked dimer mutants, A444C- and L451C-SR-BI, exhibited normal selective uptake of [ 3 H]-cholesteryl oleyl ether, despite slightly reduced [ 125 I]-HDL binding. SR-BI-mediated cholesterol efflux to HDL from cells pre-labeled with [ 3 H]-cholesterol was also unaltered by the presence of locked dimers. Finally, we investigated the ability of WT and mutant SR-BI receptors to alter accessibility of membrane free cholesterol to exogenous cholesterol oxidase (as judged by cholestenone levels). L451C- or G453C-SR-BI expression led to reduced cholestenone production compared to WT-SR-BI, suggesting that these mutants may be defective in reorganizing pools of membrane cholesterol. In conclusion, our preliminary data suggest that limiting conformational flexibility between TMDs by forcing locked dimers of SR-BI may not have a major impact on SR-BI-mediated cholesterol transport. However, locked dimers of SR-BI appear to affect the ability of SR-BI to modulate plasma membrane pools of free cholesterol, and this deserves further investigation.

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Photometric determination of free cholesterol via cholesterol oxidase and carbon nanotube supported Prussian blue as a peroxidase mimic

Microchimica Acta ◽

10.1007/s00604-017-2235-y ◽

2017 ◽

Vol 184 (7) ◽

pp. 2181-2189 ◽

Cited By ~ 43

Author(s):

Yanfang He ◽

Xiangheng Niu ◽

Libo Shi ◽

Hongli Zhao ◽

Xin Li ◽

...

Keyword(s):

Carbon Nanotube ◽

Prussian Blue ◽

Free Cholesterol ◽

Cholesterol Oxidase ◽

Photometric Determination ◽

Peroxidase Mimic

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Fluorometric Enzymatic Determination of Total Cholesterol in Serum

Clinical Chemistry ◽

10.1093/clinchem/21.11.1605 ◽

1975 ◽

Vol 21 (11) ◽

pp. 1605-1608 ◽

Cited By ~ 75

Author(s):

Hua-shan Huang ◽

Jui-chang W Kuan ◽

George G Guilbault

Keyword(s):

Serum Cholesterol ◽

Free Cholesterol ◽

Cholesterol Oxidase ◽

Correlation Coefficients ◽

Total Serum ◽

Total Serum Cholesterol ◽

Enzymatic Method ◽

Enzymatic Determination ◽

Production Of Hydrogen

Abstract We describe a fluorometric enzymatic method for determining total serum cholesterol, based on hydrolysis of cholesterol esters to free cholesterol by cholesterol ester hydrolase (EC 3.1.1.13). The free cholesterol formed, as well as that initially present, is then oxidized by cholesterol oxidase (EC 1.1.3.6) to cholest-4-en-3-one with simultaneous production of hydrogen peroxide. The latter catalytically oxidizes homovanillic acid in the presence of peroxidase (EC 1.11.1.7) to form the highly fluorescent 2,2'-dihydroxy-3,3'-dimethoxy-biphenyl-5,5'-diacetic acid. A calibration curve is constructed from data on a series of standard cholesterol solutions vs. the corresponding fluorescence change (Δf/5 min). This curve is linear up to 4.0 g of total serum cholesterol per liter of serum. The method is specific, precise, accurate, rapid, and simple, and results correlate well with those obtained by both the Liebermann-Burchard procedure and the colorimetric enzymatic method (correlation coefficients, 0.984 and 0.981, respectively)

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