Automated enzymic measurement of total cholesterol in serum.

1978 ◽  
Vol 24 (1) ◽  
pp. 108-114 ◽  
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.

One-minute electrochemical enzymic assay for cholesterol in biological materials.

1981 ◽  
Vol 27 (12) ◽  
pp. 1978-1982 ◽  
Author(s):  
L C Clark ◽  
C A Duggan ◽  
T A Grooms ◽  
L M Hart ◽  
M E Moore
Keyword(s):  
Hydrogen Peroxide ◽  
Cholesterol Oxidase ◽  
Density Lipoprotein ◽  
Cholesterol Esters ◽  
Unesterified Cholesterol ◽  
Polycarbonate Membrane ◽  
Platinum Anode ◽  
Tissue Homogenates ◽  
Enzymic Assay ◽  
Kendall Method

Abstract In this rapid and specific micro-scale electrochemical enzymic assay for cholesterol and cholesterol esters, 10 microL of standard or sample is injected directly into a heated (50 degrees C) thermostated, oxystated cuvet containing pH 7.25 buffer, cholesterol oxidase (EC 1.1.3.6), and cholesterol esterase (EC 3.1.1.13). The cholesterol esters are hydrolyzed by the esterase, and the cholesterol is simultaneously oxidized by the oxidase. The hydrogen peroxide produced from oxidation of the unesterified cholesterol is measured by a polarographic anode covered with an acetate/polycarbonate membrane. The membrane allows hydrogen peroxide to diffuse to the platinum anode, where it is oxidized, but prevents the diffusion of ascorbic acid, uric acid, and bilirubin to the electroactive surface. Turbidity does not interfere. The correlation (r) between results by our method and the Abell-Kendall method for 105 samples of serum was 0.9994 and for 105 samples of plasma was 0.9997. Our method is convenient for the analysis of high-density lipoprotein cholesterol in plasma and serum supernates and in many kinds of tissue homogenates. Its limitations are also described.

scholarly journals Filipin vs enzymatic localization of cholesterol in guinea pig, mink, and mallard duck testicular cells.

1994 ◽  
Vol 42 (12) ◽  
pp. 1539-1554 ◽  
Author(s):  
R M Pelletier ◽  
M L Vitale
Keyword(s):  
Guinea Pig ◽  
Lipid Droplets ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
False Negative ◽  
Cholesterol Esters ◽  
Enzymatic Method ◽  
Mallard Duck ◽  
Cholesterol Levels ◽  
Testicular Cells

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.

Preparation and Properties of a Cholesterol Oxidase from Nocardia sp. and Its Application to the Enzymatic Assay of Total Cholesterol in Serum

1973 ◽  
Vol 19 (12) ◽  
pp. 1350-1356 ◽  
Author(s):  
W Richmond
Keyword(s):  
Hydrogen Peroxide ◽  
Total Cholesterol ◽  
Enzyme Assay ◽  
Cholesterol Oxidase ◽  
Xylenol Orange ◽  
Extraction And Purification ◽  
Production Of Hydrogen ◽  
Nocardia Sp ◽  
Wide Ph Range ◽  
Ph Range

Abstract I describe the characterization, extraction, and purification of a cholesterol:oxygen oxidoreductase (EC 1.1.3.6) from Nocardia sp. This enzyme catalyzes oxidation of cholesterol to Δ4-choIestenone, with production of hydrogen peroxide. It is very stable, active over a wide pH range, and has a Km of 1.4 x 10-5 mol/ liter. It is highly specific for Δ4- or Δ5-3β-hydroxycholestanes, and may be applied to the assay of serum total cholesterol. In the procedure presented here, hydrogen peroxide is measured by reaction with quadrivalent titanium and xylenol orange. This constitutes a one-enzyme assay with stable reagents, which does not require protein precipitation and is not subject to interference from hemoglobin or bilirubin.

scholarly journals Oxidase Determination of Plasma Cholesterol as Cholest-4-En-3-One Using Iso-Octane Extraction

1981 ◽  
Vol 18 (2) ◽  
pp. 64-70 ◽  
Author(s):  
Paul Trinder
Keyword(s):  
Hydrogen Peroxide ◽  
Plasma Cholesterol ◽  
Cholesterol Oxidase ◽  
Cholesterol Esters ◽  
Ethanolic Solution ◽  
Single Determination

Cholesterol esters in 20 μl of plasma are hydrolysed with hot ethanolic KOH. Preformed cholesterol and cholesterol released by hydrolysis is reacted with cholesterol oxidase to form hydrogen peroxide and cholest-4-en-3-one, which is extracted from alkaline 50% v/v ethanolic solution with iso-octane. The absorbance of the ketone in iso-octane at the 232 nm peak is used to measure the cholesterol originally present. Plasma blanks obtained by omitting the cholesterol oxidase from the reagent show negligible absorbance even when the samples are grossly icteric, lipaemic, or haemolysed. The test is carried out in a single glass screw-capped tube, and the absorbance given by a sample containing 6·25 mmol/l cholesterol is ≏ 0·44, corresponding to a molar absorbance of ≏ 17 500. The conversion of cholesterol to cholest-4-en-3-one is stoichiometric, and the absorbance of the iso-octane layer is stable for at least 48 hours. A single determination occupies 30 minutes; 30 samples can be analysed in 1½ hours.

Aspects of hepatic cholesterol metabolism in normal and dystrophic chicken embryos

1983 ◽  
Vol 61 (6) ◽  
pp. 378-386 ◽  
Author(s):  
D. E. Kuhn ◽  
D. M. Logan
Keyword(s):  
Total Cholesterol ◽  
Free Cholesterol ◽  
Cholesterol Metabolism ◽  
Cholesterol Content ◽  
Normal Embryo ◽  
Total Protein Content ◽  
Cholesterol Esters ◽  
Hepatic Cholesterol ◽  
Chicken Embryos ◽  
Dystrophic Chicken

Several aspects of hepatic cholesterol metabolism have been studied in normal and dystrophic chicken embryos (12 days in ovo). Dystrophic embryo livers weigh the same and have the same total protein content as their normal counterparts. However, the total cholesterol content of dystrophic embryo livers is significantly decreased compared with the content of normal embryo livers. This decrease in total cholesterol is due mainly to a large decrease in the amount of cholesterol esters, although the free cholesterol content is also decreased. The proportions of free cholesterol and cholesterol esters are 43 and 57%, respectively, in normal embryo livers, but this proportion is essentially reversed in dystrophic embryo livers (i.e., 56 and 44%). The decreased total cholesterol content of dystrophic embryo livers is not apparently due to a decrease in the rate of free cholesterol de novo biosynthesis or to a decrease in the rate of free cholesterol esterification. However, the decrease in cholesterol ester content may be due to an increase in the rate of its hydrolysis, as a result of increased levels of sterol ester hydrolase in dystrophic livers. These data are consistent with the proposal that membranes in dystrophic tissues are altered as a result of altered cholesterol metabolism and utilization.

Determination of Total and Esterified Cholesterol in Serum

1966 ◽  
Vol 12 (11) ◽  
pp. 739-747 ◽  
Author(s):  
Charles Sobel ◽  
Alberto Fernandez
Keyword(s):  
Total Cholesterol ◽  
Free Cholesterol ◽  
Serum Lipids ◽  
Solvent Mixture ◽  
Solvent Composition ◽  
Cholesterol Esters ◽  
Simple Method ◽  
Reference Procedure

Abstract A simple method for the determination of total and esterified cholesterol is described. Serum lipids are extracted into a solvent mixture and aliquots of the extract are analyzed directly for free and total cholesterol by the Zlatkis reaction. Esterified cholesterol is calculated by difference. Free cholesterol is determined under conditions of temperature and solvent composition such that the cholesterol esters do not react. The method yields quantitative recoveries of free and esterified cholesterol from serum. Data are presented on the precision of the method, and on results of a comparison with the reference procedure of Sperry and Webb.

Use of cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis.

1976 ◽  
Vol 22 (10) ◽  
pp. 1579-1588 ◽  
Author(s):  
W Richmond
Keyword(s):  
Total Cholesterol ◽  
Continuous Flow ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
Xylenol Orange ◽  
Flow Analysis ◽  
Dual Channel ◽  
Aqueous Micellar Solution ◽  
Continuous Flow Analysis ◽  
Triton X

Abstract Idescribe an assay for total cholesterol in serum, with use of the AutoAnalyzer ii (Technicon), in which cholesterol esters are saponified by alkali, cholesterol is held in aqueous micellar solution with a surfactant (Triton X-100) and oxidized by cholesterol oxidase, and the hydrogen peroxide produced is measured by chelation with Ti4+ and xylenol orange. An assay for free cholesterol in serum, based on similar principles, is also described, and the two can be run simultaneously on a dual-channel AutoAnalyzer II. Standard solutions of cholesterol in isopropanol have poorer carryover characteristics than sera, and therefore do not reach the same continuous-flow steady state as sera of equivalent concentrations. Consequent potential calibration errors are avoided by using micellar solutions of cholesterol containing albumin for standardization. The formation of cholesterol peroxide in solutions of cholesterol in isopropanol is demonstrated, and this constitutes another potential source of error in the calibration of enzymic cholesterol assays. In analyzing patients' sera, results of the total cholesterol assay correlate well with those of a mechanized method in which cholesterol esterase and cholesterol oxidase are used; an automated Abell method, calibrated with solutions of cholesterol in isopropanol, gave slightly higher values. Determinations of the ratio of free cholesterol to total cholesterol by our automated cholesterol exidase assays given values that agree well with published results in which digitonin precipitation is used.

Cholesterol oxidase-based determination, by continuous-flow analysis, of total and free cholesterol in serum.

1976 ◽  
Vol 22 (10) ◽  
pp. 1627-1630 ◽  
Author(s):  
R F Lie ◽  
J M Schmitz ◽  
K J Pierre ◽  
N Gochman
Keyword(s):  
Total Cholesterol ◽  
Continuous Flow ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
Flow Analysis ◽  
Colorimetric Measurement ◽  
Continuous Flow Analysis ◽  
Hydrolysis Of ◽  
Automated Determination

Abstract We describe a continuous-flow, automated determination of total cholesterol in serum, which is based on enzymatic hydrolysis of cholesterol esters, oxidation of cholesterol by cholesterol oxidase, and colorimetric measurement of liberated perioxide with 4-aminoantipyrine, phenol, and peroxidase. Free cholesterol is determined with the same AutoAnalyzer II manifold and reagents, except that cholesterol esterase is omitted from the reagent. Cholesterol-in-serum materials that have been assayed by an established method are used for calibration. We found this approach to be necessary because primary cholesterol standards in organic solvents are incompatible with the aqueous reagent. Results of the enzymatic total cholesterol method correlated well with those by an AutoAnalyzer II method which involves an extraction with isopropanol and the Liebermann-Burchard color reaction (total cholesterol, g/liter, yenz= 0991xlb +0.05;r=0.996). Results of the enzymatic free cholesterol procedure agreed satisfactorily with one in which free cholesterol is precipitated as the digitonide and subsequently analyzed colorimetrically with the Liebermann-Burchard reaction (free cholesterol, %, yenz = 0.982xdig -0.7;r= 0.956).

Enzymic determination of the free cholesterol fraction of high-density lipoprotein in plasma with use of 2,4,6-tribromo-3-hydroxybenzoic acid.

1988 ◽  
Vol 34 (9) ◽  
pp. 1799-1804 ◽  
Author(s):  
J S Moshides
Keyword(s):  
High Density Lipoprotein ◽  
Total Cholesterol ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
Density Lipoprotein ◽  
High Density ◽  
Hydroxybenzoic Acid ◽  
Automated Method ◽  
Cholesterol Fraction

Abstract A highly sensitive enzymic colorimetric reagent is described for determination of the free cholesterol fraction of high-density lipoprotein (HDL), which represents about 20% of the total cholesterol content of this lipoprotein. For greater sensitivity with respect to cholesterol, I used 2,4,6-tribromo-3-hydroxybenzoic acid instead of phenol in the cholesterol oxidase/peroxidase/4-aminoantipyrine reagent system. This allows determination of the free cholesterol fraction of HDL isolates prepared with polyethylene glycol 6000, a method for precipitating beta-lipoprotein that involves a twofold dilution of plasma. The reagent, adapted for use with a Cobas-Bio centrifugal analyzer, results in between-run and within-run CVs of less than 3% and a linearity to at least 400 mg of HDL free cholesterol per liter. Comparison with results by Trinder's cholesterol method, which measures cholest-4-en-3-one at 232 nm, showed good correlation (r = 0.9829, slope 1.0001, and y-intercept +2.4797 mg/L). With the manual procedure for HDL free cholesterol, between-batch and within-batch CVs were less than 5%, and results correlated well with those by the automated method (r = 0.9975, slope 0.9839, and y-intercept +2.4327 mg/L). The mean (and SD) HDL free cholesterol for 123 men was 96.8 (30.6) mg/L and for 122 women 136.4 (36.8) mg/L, indicating a distinct sex-related difference, similar to that found for HDL total cholesterol. HDL free cholesterol in plasma may therefore be a potential new predictor of coronary heart disease.

scholarly journals Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1966 ◽  
Vol 101 (3) ◽  
pp. 819-830 ◽  
Author(s):  
PR Raggatt ◽  
MW Whitehouse
Keyword(s):  
Steroid Hormones ◽  
Adrenal Cortex ◽  
Free Cholesterol ◽  
Cholesterol Oxidase ◽  
Enoic Acid ◽  
Hydroxyl Group ◽  
Cholesterol Oxidation ◽  
Cholesterol Esters ◽  
Cholesteryl Acetate

1. Cholesteryl 3beta-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K(m) for cholesteryl sulphate is 500mum and for free cholesterol 50mum under the same conditions. 3. Cholesteryl 3beta-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K(i) for cholesteryl phosphate 28mum, for cholesteryl sulphate 110mum, for cholesteryl acetate 65mum) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20alpha-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K(i) for pregnenolone is 130mum and K(i) for 20alpha-hydroxycholesterol is 17mum. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K(i)16mum). A number of other Delta(5)-3beta-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3beta-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20alpha-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3beta-Hydroxychol-5-enoic acid, 3alpha-hydroxy-5beta-cholanic acid and 3beta-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3beta-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.

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