Fluorometric enzymatic determination of total cholesterol in serum.

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 (deltaf/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).     

Simple enzymatic determination of total cholesterol in gallstones

In this simple method for rapid enzymatic determination of total cholesterol in human gallstones, gallstone powder is dissolved in an 80/20 by vol mixture of N,N-dimethylformamide and dimethyl sulfoxide and reacted directly with the aqueous enzymatic reagent, without further treatment. The organic solvents do not interfere with enzymatic activities of cholesterol oxidase, esterase, and peroxidase in the assay mixture. The method is reproducible (CVs 3.7% to 6.6%), analytical recoveries ranged from 98.6% to 102%, and linearity is good. Furthermore, results correlated well with those obtained by infrared spectroscopic measurements.     

Automated method for the direct determination of total serum cholesterol

An automated method for determining total cholesterol in serum is described which does not require a preliminary solvent extraction. A special glass unit has been constructed and details of the preparation of cholesterol acetate standards in acetic acid are given. Thirty samples, including five standards, can be estimated per hour and the volume of sample required is 0.5 ml.     

Implementation of cellulomonas cholesterol oxidase for total serum cholesterol determination by the endpoint method

Cellulomonas has been shown to be a good source of cholesterol oxidase in addition to Streptomyces for serum cholesterol determination by the endpoint method, inexpensive in cost, and showing excellent performance. For clinical use, we have assessed the reliability of Cellulomonas reagent for cholesterol determination. We constructed the user-defined endpoint methods on three automated analyzers. The analytical performances (linearity, precision, recovery, interference, stability, and comparison with the standardized method) of Cellulomonas cholesterol reagents were evaluated and compared to those of Streptomyces reagents. Linearity (18.1-23.3 mmol/L) and stability of reagents (6-11 weeks) depended on the analyzers being used. The average within-run and between-day % coefficients of variation (CVs) ranged from 1.44 to 2.45 and 1.98 to 2.99, respectively, and were within National Cholesterol Education Program analytical criteria (相似文献   

Performance of four sources of cholesterol oxidase for serum cholesterol determination by the enzymatic endpoint method

BACKGROUND: Cholesterol oxidase is used for the determination of serum cholesterol. It can be derived from Streptomyces, Pseudomonas fluorescens, Cellulomonas, and Brevibacterium. This study compared the performance characteristics of four enzymes in the endpoint cholesterol determination. METHODS: Using the Mega analyzer, we studied assay optimization, linearity, precision, recovery, interference, stability, and compared 110 patient samples. RESULTS: The linearity for the four enzymes was up to 13.0 mmol/l at the optimal enzyme activity. The average within-run CVs ranged from 1.6% to 1.9% and between-day ranged from 2.8% to 3.0%, within the NCEP analytical criteria. The analytical recoveries obtained from four reagents ( approximately 96.5%) were excellent. The assays using these enzyme sources compared favorably with the commercial method and appeared accurate near the clinical decision cut-points. Hemoglobin concentration at 1.9 g/l interfered with the P. fluorescens cholesterol oxidase. Bilirubin caused a negative interference while lipemia generated a positive interference with all enzyme sources. Reagents were stable up to 6 weeks. CONCLUSIONS: Streptomyces, Cellulomonas, and Brevibacterium were essentially analytically equivalent. Streptomyces and Cellulomonas cholesterol oxidase are one-quarter as expensive Brevibacterium. Cellulomonas is a new source of cholesterol oxidase for determining serum cholesterol by the endpoint method.     

Application of Streptomyces and Brevibacterium cholesterol oxidase for total serum cholesterol assay by the enzymatic kinetic method

BACKGROUND: Using non-esterified cholesterol standard, Brevibacterium and Streptomyces are found as suitable sources of cholesterol oxidase for kinetic cholesterol assay. For clinical use, we investigated the suitability of these enzymes for cholesterol determination in human serum. METHODS: We compared the performance of reagents containing 2 enzymes for the kinetic determination of total serum cholesterol with the standardized endpoint method. RESULTS: Reagent containing Streptomyces enzyme was more sensitive than that of Brevibacterium, with linearity up to 20.7 and 2.6 mmol/l, respectively. The analytical reaction for Streptomyces showed a shorter lag phase (148 s) and a steeper slope (absorbance vs. time) than that of Brevibacterium (246 s). The assay using Streptomyces reagent was precise and accurate and compared favorably with the endpoint method (y=1.06x-0.15, r=0.996, bias=0.21 mmol/l). Hemoglobin as high as 7.5 g/l did not interfere while turbidity greater than 2+ (absorbance >0.778 at 670 nm) and bilirubin concentrations >171.0 micromol/l did interfere (in a negative interference). Reagent was stable up to at least 8 weeks. CONCLUSIONS: The Streptomyces cholesterol oxidase, with 3,4-dichlorophenol, proved a suitable source for serum total cholesterol determination by the kinetic method.     

Kinetic enzymic method for automated determination of total cholesterol in serum

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.     

生化试剂对循环酶法测定血清总胆汁酸的干扰原因

目的探讨生化试剂对循环酶法测定血清总胆汁酸(TBA)的干扰原因,探寻消除干扰的办法。方法以门冬氨酸氨基转移酶、碱性磷酸酶、钙等20个生化常规项目试剂作用作为观察对象,确认对TBA具有显著干扰的生化试剂,且经过设定特殊程序减小仪器的携带污染。结果测定混合血清当中20份TBA水平,其平均值为(8.16±0.012)μmol/L;在20个观察项目当中,尿酸试剂(UA)对于测定总胆汁酸(TBA)结果影响差异具有统计学意义(P0.05),存在系统误差;总胆固醇(CHOL)、三酰甘油(TG)项目试剂对于测定TBA结果影响差异具有统计学意义(P0.05),对于TBA测定具有明显干扰。结论全自动化生化分析仪测定TBA时存在项目之间的互相污染,因此设定有效、合理的特殊冲洗程序能够有效地对消灭携带污染对TBA测定干扰。     

Ezymatic method for the automated determination of total serum cholesterol (author's transl)]

A simple kinetic method for the automated determination of total serum cholesterol was developed using cholesterol esterase, cholesterol oxidase, catalase and the color reaction according to Hantzsch. The procedure has been adapted to the Braun SysteMatik, Greiner GSA II, Eppendorf 5032, Lars Ljungberg Autolab, Perkin-Elmer C4 and Perkin-Elmer C4B analyzers. The variants of the method yielded satisfactory results with regard of precision, accuracy and sensitivity to interferences.     

Amperometric determination of total cholesterol in serum, with use of immobilized cholesterol ester hydrolase and cholesterol oxidase.

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 enzyme layer easily, thus catalyzing the enzymatic transformation quickly. Hydrogen peroxide resulting from a catalytic reactions was measured amperometrically at +0.60 V cs. 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 700 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.     

Enzymic determination of total serum cholesterol with centrifugal analyzers (author's transl)]

The enzymic determination of total cholesterol described by P. R?schlau (Z. Klin. Chem. Klin. Biochem. 12, 403-407 (1974)) was modified and adapted to the Centrifichem system. The method which requires only 5 mul serum, is accurate, quick and simple. A separate serum blank is not necessary. Compared with the manual method, smaller quantities of reagents used and the resulting decrease in cost are appreciable for large series of analyses. The course of the reaction at 30 and 37 degrees C, the linearity, calibration, precision, recovery, and correlation with the Lieberman-Burchard method and the enzymic manual method are described.