Direct Fluorometric Determination of Total Cholesterol in Serum Using Derivatized Cholesterol Oxidase

2000 ◽  
Vol 54 (8) ◽  
pp. 1157-1162 ◽  
Author(s):  
Javier Galbán ◽  
José F. Sierra ◽  
José M. López Sebastian ◽  
Susana de Marcos ◽  
Juan R. Castillo
Keyword(s):  
Blood Serum ◽  
Total Cholesterol ◽  
Cholesterol Oxidase ◽  
Enzymatic Reaction ◽  
Direct Determination ◽  
Analytical Signal ◽  
Cholesterol Concentration ◽  
Serum Samples ◽  
Response Range

In this paper the use of cholesterol oxidase derivatized with a fluorescein derivative is proposed for the direct determination of total cholesterol in blood serum. The method is based on the changes in the fluorescence of the solution during the enzymatic reaction (λexe = 498 nm and λem 519 nm). A mathematical model which relates the analytical signal to the total cholesterol concentration was developed, and the model can also be used to obtain some of the thermodynamic constants of the process. The method has a linear response range up to 70 mg/L of cholesterol, a detection limit of 2.5 mg/L, and the precision was 1.0% (40 mg/L cholesterol, n = 10). The method was applied to total cholesterol determination in blood serum samples. The results were compared to those obtained by a commercial analyzer, and statistically similar results were obtained. The use of derivatized cholesterol oxidase makes it possible to simplify the methodology normally used in this type of determination (the indicator reaction is avoided and the number of reagents reduced), with the added advantage that the analytical signal is independent of the concentrations of O2 and cholesterol oxidase.

Kinetic enzymic method for automated determination of total cholesterol in serum.

1983 ◽  
Vol 29 (10) ◽  
pp. 1798-1802 ◽  
Author(s):  
R Deeg ◽  
J Ziegenhorn
Keyword(s):  
Total Cholesterol ◽  
Incubation Temperature ◽  
Cholesterol Oxidase ◽  
Fixed Time ◽  
Competitive Inhibitor ◽  
Cholesterol Concentration ◽  
Total Analysis ◽  
Single Standard ◽  
Automated Determination

Abstract We describe a rapid, kinetic, fixed-time method for determining serum total cholesterol by use of cholesterol esterase, cholesterol oxidase, and the indicator reaction with peroxidase, 4-aminophenazone, and phenol. On addition of the competitive inhibitor 3,4-dichlorophenol the Michaelis constant of cholesterol oxidase is apparently increased, which extends the linear relation between absorbance change and cholesterol concentration to 20.7 to 25.9 mmol/L, depending on the analyzer being used. For calibration, a single standard is used. Total analysis time is in the range of 80 to 210 s. Incubation temperature is 25 degrees C or 37 degrees C. The single-reagent procedure has been adapted to three different centrifugal analyzers and to the Eppendorf ACP 5040 analyzer. It yields precise and accurate results and is insensitive to potential interferences.

Determination of urea in 10-?l blood serum samples with a urease reactor / ion-selective electrode cell

1983 ◽  
Vol 81 (1-2) ◽  
pp. 135-147 ◽  
Author(s):  
U. Thanei-Wyss ◽  
W. E. Morf ◽  
P. Lienemann ◽  
Z. Stefanac ◽  
I. Mostert ◽  
...  
Keyword(s):  
Blood Serum ◽  
Ion Selective Electrode ◽  
Selective Electrode ◽  
Serum Samples ◽  
Electrode Cell

Comparison of the Du Pont aca and Dow methods for determination of high-density lipoprotein cholesterol.

1984 ◽  
Vol 30 (1) ◽  
pp. 127-129 ◽  
Author(s):  
N N Rehak ◽  
R J Elin ◽  
R Chesler ◽  
E Johnson
Keyword(s):  
High Density Lipoprotein ◽  
Disease Control ◽  
Total Cholesterol ◽  
High Density Lipoprotein Cholesterol ◽  
Dextran Sulfate ◽  
Density Lipoprotein ◽  
High Density ◽  
Lipoprotein Cholesterol ◽  
Serum Samples

Abstract We compared the Du Pont aca (phosphotungstate-enzymic cholesterol) and the Dow (dextran sulfate/Mg2+-enzymic cholesterol) methods for the determination of high-density lipoprotein cholesterol (HDLC) and total cholesterol in serum from 113 patients. The aca results for both total cholesterol and HDLC were significantly greater (p less than 0.0001) than the Dow results, the aca method overestimating the HDLC concentration (mean recovery 107.2% in serum samples with values assigned by the Centers for Disease Control). The precision of the aca method for HDLC was essentially the same as that of the Dow method. Bilirubin (up to 0.17 g/L), hemoglobin (up to 4 g/L), and slight lipemia (triglycerides up to 5.4 g/L) did not interfere with the aca method.

scholarly journals Determination of ionized magnesium in platelets and correlation with selected variables

1996 ◽  
Vol 42 (5) ◽  
pp. 744-748 ◽  
Author(s):  
J E Niemela ◽  
B M Snader ◽  
R J Elin
Keyword(s):  
Total Cholesterol ◽  
Inverse Correlation ◽  
Reference Interval ◽  
Cholesterol Concentration ◽  
Magnesium Concentration ◽  
Serum Electrolytes ◽  
Ionized Magnesium ◽  
And Gender ◽  
Dissociation Constant Kd

Abstract We describe a method for determining the intracellular ionized magnesium concentration ([Mg2+]i) in platelets by using the fluorescent probe FURAPTRA. We determined the dissociation constant (KD) of FURAPTRA for Mg2+ (2.26 +/- 0.29 mmol/L), within-day assay variability (CV = 6.8%), among-day intraindividual variability (CV = 11.0%), variability after a 4-h delay in processing the blood specimen (t = 1.2, P >0.2; F = 6.2, P <0.02), and the reference interval (0.23-0.59 mmol/L) for this assay. We also evaluated the correlation between platelet [Mg2+]i and concentrations of selected serum electrolytes, proteins, and total cholesterol; age; body mass index; and gender. Only the inverse correlation between platelet [Mg2+]i and serum total cholesterol concentration in men was significant (r=-0.66, P <0.005).

Improvement of enzyme carbon paste-based biosensor using carbon nanotubes for determination of water-soluble analogue of vitamin E

2015 ◽  
Vol 69 (1) ◽  
Author(s):  
Milan Sýs ◽  
Radovan Metelka ◽  
Tomáš Mikysek ◽  
Karel Vytřas
Keyword(s):  
Carbon Nanotubes ◽  
Vitamin E ◽  
Electrochemical Techniques ◽  
Enzymatic Reaction ◽  
Direct Determination ◽  
Water Soluble ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Total Phenolic ◽  
Antioxidant Power

AbstractThe catalytic oxidation of a synthetic water-soluble analogue of vitamin E (α-tocopherol, Trolox) by tyrosinase enzyme in the presence of molecular oxygen was studied using electrochemical techniques. This specific enzymatic reaction was exploited for the preparation of a biosensor based on the amperometric reduction of the electroactive product (α-tocoquinone) formed. An electroactive surface of the transducers used was covered with a thin conductive layer of Nafion containing tyrosinase. Significant progress in sensitivity towards polyphenolic compounds such as Trolox was achieved at CPE with carbon nanotubes immobilised on its surface (CPE/CNTs) as electric transducers. The biosensor so developed can be used for the direct determination of total phenolic content (TPC). This important nutrition value can be expressed as the mass equivalent of Trolox, i.e. Trolox equivalent antioxidant capacity (TEAC), which could be used as an alternative to the evaluations currently used based on spectrophotometric methods such as total radical-trapping antioxidant parameter (TRAP), ferric reducing-antioxidant power (FRAP) or 1,1-diphenyl-2-picrylhydrazyl spectrometric assay (DPPH). The effects of the enzyme amount in the Nafion layer (3.0 μg), the influence of the nanoparticles present, the optimal pH value suitable for enzymatic activity (7.0), and the kinetics of enzymatic and electrochemical reactions were studied using cyclic voltammetry (CV). The determination of optimal conditions for amperometry in batch configuration (working potential, speed of stirring, volume of sample, calibration curve, etc.) was not a target of this electrochemical study.

Effects of sample aging on total cholesterol values determined by the automated ferric chloride-sulfuric acid and Liebermann-Burchard procedures.

1980 ◽  
Vol 26 (5) ◽  
pp. 592-597 ◽  
Author(s):  
P D Wood ◽  
P S Bachorik ◽  
J J Albers ◽  
C C Stewart ◽  
C Winn ◽  
...  
Keyword(s):  
Sulfuric Acid ◽  
Total Cholesterol ◽  
Ferric Chloride ◽  
Continuous Flow ◽  
Frozen Storage ◽  
Cholesterol Concentration ◽  
Manual Method ◽  
Fresh Plasma ◽  
Sulfuric Acid Method

Abstract To investigate the comparability of three commonly used methods for determination of total cholesterol in plasma in several studies, we used fresh plasma samples as well as plasmas and reference sera that had been stored frozen at −15 degrees C for as long as several years. Duplicate determinations by the manual method of Abell et al. (J. Biol. Chem. 195: 357, 1952) were compared with estimates from one to five continuous-flow analyzers by the ferric chloride-sulfuric acid procedure and also with estimates from five to 13 continuous-flow analyzers by the Liebermann-Burchard procedure with calibrator, as part of the laboratory standardization activities of the Lipid Research Clinics. The agreement among all three procedures was generally within acceptable limits (within 5% of the manual method) when plasmas or sera were fresh or had been frozen for less than one month. Results by the manual method of Abell et al. agreed well with those by the automated Liebermann-Burchard method for samples that had been stored at −15 degrees C for as long as two years. However, the automated ferric chloride-sulfuric acid procedure often showed unacceptably high values (as compared with those from the manual method) for samples that had been stored frozen for a year or more. With the ferric chloride-sulfuric acid method, measured cholesterol concentration increased about 2.5% per year of storage for at least two years. We conclude that reference sera of plasmas that have been kept in long-term frozen storage (−15 degrees C) are not suitable for ongoing standardization of the automated ferric chloride-sulfuric acid assay for cholesterol.

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