Direct Determination of Total Serum Cholesterol by On-Column Gas-Liquid Chromatographic Analysis without Previous Derivatisation Compared with WHO-CDC Reference Method

1983 ◽  
Vol 20 (6) ◽  
pp. 360-363 ◽  
Author(s):  
J W I Brunnekreeft ◽  
G J M Boerma ◽  
B Leijnse
Keyword(s):  
Serum Cholesterol ◽  
Direct Determination ◽  
Reference Method ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Gas Liquid Chromatography ◽  
Accuracy And Precision ◽  
Liquid Chromatographic Analysis ◽  
Liquid Chromatographic

A method for the direct determination of total serum cholesterol by on-column gas-liquid chromatography is described. The French reference method developed by Gambert et al.1 served as a model for our method, which is fast and less laborious than the well-known CDC reference method of Cooper et al.23 based on the Abell-Kendall technique.4 The accuracy and precision of our on-column gas-liquid chromatographic method and the CDC reference method are comparable. The obvious advantage of this proposed gas-liquid chromatographic reference method is its increased analytical speed. The problem of the specificity of our method in relation to cholestanol is discussed.

Automated Reaction-Rate Method for the Direct Determination of Total Serum Cholesterol by Use of the "CentrifiChem" Parallel Fast Analyzer

1973 ◽  
Vol 19 (8) ◽  
pp. 937-941 ◽  
Author(s):  
K A Slickers ◽  
L Edwards ◽  
J Daly ◽  
G Ertingshausen
Keyword(s):  
Serum Cholesterol ◽  
Regression Line ◽  
Direct Determination ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Triglyceride Content ◽  
Reference Serum ◽  
Rate Method ◽  
High Bilirubin

Abstract The direct procedure for determining serum cholesterol described by Wybenga et al. [Clin. Chem. 16, 980 (1970)] can be used to assay 29 samples in 10-min reaction time by using the "CentrifiChem" parallel fast analyzer (Union Carbide). Commercial reference serum was used for standardization. Correlation of results with those obtained by the method of Abell et al. [J. Biol. Chem. 195, 357 (1952)] for 54 samples yielded a regression line with a slope (m) of 0.897 and a y-intercept (b) of 231 mg/liter; the correlation coefficient (r) was 0.971. Compared with the manual procedure of Parekh and Jung [Anal. Chem. 42, 1423 (1970)] for 146 samples, the corresponding values were m = 0.991, b = 27 mg/ liter, and r = 0.981. Compared to serum extracts assayed with the "AutoAnalyzer" (Standard Technicon Methodology N-24a), 186 samples gave corresponding values of m = 0.990, b = 92 mg/ liter, and r = 0.979. No bias significant at the 95% confidence level existed versus any of the three methods. Sera containing abnormally high bilirubin concentrations (>50 mg/liter) or abnormally high triglyceride content (>5,000 mg/liter) did not give significantly different cholesterol values on the CentrifiChem as compared with values obtained with either the AutoAnalyzer or the Parekh and Jung method.

Fluorescent assay of total serum cholesterol, with use of gas-liquid chromatography to study saponification efficiency.

1977 ◽  
Vol 23 (11) ◽  
pp. 1976-1983 ◽  
Author(s):  
E J Majeski ◽  
E J Seltzer ◽  
P L Carter ◽  
D R Howlett ◽  
J D Stuart
Keyword(s):  
Liquid Chromatography ◽  
Total Cholesterol ◽  
Methylene Chloride ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Gas Liquid Chromatography ◽  
Cholesterol Esters ◽  
Accuracy And Precision ◽  
Concentrated Sulfuric Acid

Abstract We describe a fluorescent determination of total cholesterol in serum for which the accuracy and precision are comparable to that for the method of Abell-Kendall, a method of generally accepted accuracy. By the use of quality reagents and the rigorous exclusion of water, the strong fluorophor that develops on reacting concentrated sulfuric acid with cholesterol can be used to quantitatively determine the total cholesterol in serum. We used gas-liquid chromatography to monitor the extent of saponification of the cholesterol esters, because we found them to have fluorescent efficiencies that differed from that of free cholesterol. Sodium methoxide in methanol/methylene chloride (1/3 by vol) was shown by gas-liquid chromatography to very effectively saponify the cholesterol esters in serum.

Direct Determination of Total Serum Cholesterol by Use of Double-Wavelength Spectrophotometry

1975 ◽  
Vol 21 (6) ◽  
pp. 703-707 ◽  
Author(s):  
Philip B Sommers ◽  
Peter I Jatlow ◽  
David Seligson
Keyword(s):  
Total Cholesterol ◽  
Correlation Coefficient ◽  
Serum Cholesterol ◽  
Coefficient Of Variation ◽  
Direct Determination ◽  
Accurate Method ◽  
Systematic Investigation ◽  
Total Serum ◽  
Total Serum Cholesterol

Abstract We describe a simple, accurate method for direct determination of total cholesterol in serum. Systematic investigation of a previously described modified Liebermann— Burchard reagent has indicated the necessity of accounting for both bilirubin interference and decreased specificity owing to exothermia. Double-wavelength spectrophotometry was used to optically null out bilirubin as an interfering factor, whereas adding serum to the cold reagent increases its specificity for the cholesterol color reaction. Comparison of 106 cholesterol values with those obtained by the procedure of Abell et al. [J. Biol. Chem. 195, 357 (1952)] yielded a correlation coefficient greater than 0.99; our inter-run coefficient of variation for pooled laboratory serum was 1.7%.

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.

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

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.

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.

Direct determination of the linoleate/oleate ratio in serum cholesterol esters by liquid chromatography.

1982 ◽  
Vol 28 (4) ◽  
pp. 676-680 ◽  
Author(s):  
J T Bernert ◽  
J R Akins ◽  
D T Miller
Keyword(s):  
Liquid Chromatography ◽  
Serum Cholesterol ◽  
Methyl Esters ◽  
Direct Determination ◽  
Reversed Phase ◽  
Cholesterol Esters ◽  
Serum Samples ◽  
Chromatographic Procedure ◽  
Liquid Chromatographic

Abstract We describe a convenient method for the direct determination of the serum cholesterol linoleate/cholesterol oleate (L/O) ratio by reversed-phase "high-performance" liquid chromatography. After removal of phospholipids by silicic acid chromatography, a serum extract is analyzed on a 5-micrometers particle size Ultrasphere-ODS column, eluted isocratically with acetonitrile/isopropanol (30/70, by vol). Detection is at 200 nm. Cholesterol palmitoleate interferes with the measurement when the analysis is based on peak area, but not when peak height is used. The overall precision of L/O measurements by this method was very similar to that observed with a gas-liquid chromatographic procedure, in which the cholesterol esters are first isolated and transesterified to the methyl esters. In both cases, the within-run CV for six replicate analyses was less than 2%. Analysis of 53 human serum samples by both methods yielded very similar L/O ratios. A plot of the data (our method = y) vs the usual gas-liquid chromatographic procedure gave a correlation coefficient of 0.988 and a regression equation of y = 1.03x + 0.013. Furthermore, direct analysis of serum cholesterol ester L/O ratios by our liquid-chromatographic method is simpler, quicker, and more readily adaptable to automation.

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