Fluorometric Enzymatic Determination of Total Cholesterol in Serum

1975 ◽  
Vol 21 (11) ◽  
pp. 1605-1608 ◽  
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)

Enzymatic Determination of Total Serum Cholesterol

1974 ◽  
Vol 20 (4) ◽  
pp. 470-475 ◽  
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.

Ames Award Lecture 1972. An Investigation of the Determination of Serum Cholesterol by an Enzymatic Method

1973 ◽  
Vol 10 (1-6) ◽  
pp. 79-84 ◽  
Author(s):  
Heather M. Flegg
Keyword(s):  
Serum Cholesterol ◽  
Enzymatic Assay ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Enzymatic Method ◽  
Enzymatic Determination ◽  
Cholesterol Determination

The enzymatic determination of total serum cholesterol is described. The source of the enzyme cholesterol dehydrogenase is the bacterium Nocardia erythropolis. A comparison is made between the enzymatic assay and an automated Liebermann-Burchard serum cholesterol determination.

Continuous-flow enzymatic determination of total serum cholesterol and method standardization with CDC-calibrated pooled sera.

1980 ◽  
Vol 26 (7) ◽  
pp. 896-902
Author(s):  
M A MacAulay ◽  
C L Jacklyn ◽  
J M Mathers ◽  
V A Storm
Keyword(s):  
Serum Cholesterol ◽  
Continuous Flow ◽  
Cholesterol Content ◽  
Comparison Method ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Enzymatic Method ◽  
Factors Affecting ◽  
Assigned Value

Abstract We compared Boehringer Mannheim's enzymatic kit for the continuous-flow (AutoAnalyzer II) determination of serum cholesterol with Technicon's N-24a extraction method. Results for patients' samples analyzed by the enzymatic method were higher than those by the comparison method. To evaluate accuracy in the cholesterol determinations, we enrolled the enzymatic method into the Center for Disease Control's (CDC) Lipid Standardization program. We calibrated the method by use of a pooled sera for which cholesterol content was assigned by CDC after analysis by their reference Abell-Kendall procedure. We discuss the difficulties with available calibration material and limitations in the application of some commercial control materials to the enzymatic cholesterol method. The continuous-flow variables, Michaelis-Menton constants, percent ester-hydrolase activity, and other factors affecting the performance of the enzyme-linked cholesterol method are evaluated. We believe pooled sera with an assigned value for cholesterol content is the best calibrator material.

Continuous-flow enzymatic determination of total serum cholesterol and method standardization with CDC-calibrated pooled sera.

1980 ◽  
Vol 26 (7) ◽  
pp. 896-902 ◽  
Author(s):  
M A MacAulay ◽  
C L Jacklyn ◽  
J M Mathers ◽  
V A Storm
Keyword(s):  
Serum Cholesterol ◽  
Continuous Flow ◽  
Cholesterol Content ◽  
Comparison Method ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Enzymatic Method ◽  
Factors Affecting ◽  
Assigned Value

Abstract We compared Boehringer Mannheim's enzymatic kit for the continuous-flow (AutoAnalyzer II) determination of serum cholesterol with Technicon's N-24a extraction method. Results for patients' samples analyzed by the enzymatic method were higher than those by the comparison method. To evaluate accuracy in the cholesterol determinations, we enrolled the enzymatic method into the Center for Disease Control's (CDC) Lipid Standardization program. We calibrated the method by use of a pooled sera for which cholesterol content was assigned by CDC after analysis by their reference Abell-Kendall procedure. We discuss the difficulties with available calibration material and limitations in the application of some commercial control materials to the enzymatic cholesterol method. The continuous-flow variables, Michaelis-Menton constants, percent ester-hydrolase activity, and other factors affecting the performance of the enzyme-linked cholesterol method are evaluated. We believe pooled sera with an assigned value for cholesterol content is the best calibrator material.

Amperometric Determination of Total Cholesterol in Serum, with Use of Immobilized Cholesterol Ester Hydrolase and Cholesterol Oxidase

1977 ◽  
Vol 23 (4) ◽  
pp. 671-676 ◽  
Author(s):  
Hua-shan Huang ◽  
Shia S Kuan ◽  
Guilbault George G
Keyword(s):  
Serum Cholesterol ◽  
Immobilized Enzyme ◽  
Cholesterol Oxidase ◽  
Immobilized Enzymes ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Amperometric Determination ◽  
Standard Calomel Electrode ◽  
Pass Through

Abstract We describe an electrochemical method for simple, rapid, and economical assay of total serum cholesterol with use of immobilized cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6). A rotating porous cell was specially designed to hold the immobilized enzymes firmly and to allow the reaction mixture to pass through the en¬zyme layer easily, thus catalyzing the enzymatic trans¬formation quickly. Hydrogen peroxide resulting from the catalytic reactions was measured amperometrically at +0.60 V vs. a standard calomel electrode. The calibration curve for total serum cholesterol was linear from 0 to 5.00 g/liter. The method is specific, precise, and inexpensive. Our results correlate well with those obtained by the method of Abell et al. [Stand. Methods Clin. Chem. 2, 26 (1958)] , the correlation coefficient being 0.992. Ascorbic acid or bilirubin in concentrations up to 100 mg/liter do not interfere. The immobilized enzymes are stable, and the same immobilized-enzyme stirrer can be used for at least 200 accurate, reproducible assays.

Dry film for the enzymic determination of total cholesterol in serum.

1982 ◽  
Vol 28 (5) ◽  
pp. 1159-1162 ◽  
Author(s):  
G M Dappen ◽  
P E Cumbo ◽  
C T Goodhue ◽  
S Y Lynn ◽  
C C Morganson ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cholesterol Oxidase ◽  
Comparison Method ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Gelatin Layer ◽  
Control Fluid ◽  
Hydrolysis Of ◽  
Dry Film

Abstract We have prepared a dry film for the enzymic determination of total serum cholesterol. It consists of a transparent support bearing a buffered gelatin layer, and a white reflective spreading layer that contains all of the necessary components for the detection of cholesterol. The method is based on (a) hydrolysis of cholesterol esters to cholesterol by cholesterol ester hydrolase (EC 3.1.1.13), (b) oxidation of cholesterol to cholest-4-en-4-one and hydrogen peroxide by cholesterol oxidase (EC 1.1.3.6), and (c) oxidation of a triarylimidazole leuco dye with hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) to produce a dye with maximum absorption at about 650 nm. For use over a wider range of concentration, the dye density is read at 540 nm. With reflection densitometry and appropriate mathematical transformation, readings and cholesterol concentrations are linearly related to 5500 mg/L. Results correlate well with those by the Abell-Kendall comparison method (slope 0.97, intercept 92.5, correlation coefficient 0.974, Sy.x = 250.7), and the method is precise (CV of 1.2-2.3% for a control fluid and patients' samples) and relatively free of interferences.

Turbidimetric Determination of Total Serum Cholesterol

1961 ◽  
Vol 33 (4) ◽  
pp. 561-564 ◽  
Author(s):  
G. R. Kingsley ◽  
Ozie. Robnett
Keyword(s):  
Serum Cholesterol ◽  
Total Serum ◽  
Total Serum Cholesterol
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