Application of Streptomyces and Brevibacterium cholesterol oxidase for total serum cholesterol assay by the enzymatic kinetic method
Introduction
Epidemiological and clinical studies establish serum cholesterol as a predictive association with coronary heart disease. An increased plasma cholesterol concentration leads to increased risk of developing coronary heart disease [1].
The enzymatic method for cholesterol determination can use either an endpoint or a kinetic method. The advantages of a kinetic assay are shorter analysis time, less sensitivity to interferences and no requirement of sample blank measurement [2], [3]. For measuring cholesterol by the kinetic method, Deeg and Ziegenhorn [4] demonstrated that the oxidation of cholesterol to cholest-4-ene-3-one with the simultaneous production of hydrogen peroxide of cholesterol oxidase enzyme was the rate-limiting step of whole reaction and directly proportional to cholesterol concentration. Cholesterol oxidase is derived from the several bacterium sources, such as Nocardia, Streptomyces, Mycobacteria, Brevibacterium, Schizophyllum and Pseudomonas fluorescens [4], [5], [6], [7], [8], [9]. Deeg and Ziegenhorn suggested that the enzyme isolated from Nocardia was unsuitable [4]. Its Km (1–69 × 10− 6 mol/l) was too low and the reaction did not follow first-order kinetics [7], [10], [11], [12]. Moreover, there were no competitive inhibitors that could be used to elevate the apparent Km of Nocardia [4]. They mentioned that Streptomyces was only cholesterol oxidase suitable for the kinetic method. However, 3,4-dichlorophenol, a competitive inhibitor, could be used to elevate the Km value.
Our previous study determined the kinetic properties of enzymes from Streptomyces, P. fluorescens, Brevibacterium and Cellulomonas [13]. Brevibacterium cholesterol oxidase gave the highest Km (230.3 × 10− 4 mol/l), followed by Streptomyces (2.17 × 10− 4 mol/l), P. fluorescens (0.61 × 10− 4 mol/l), and Cellulomonas (0.84 × 10− 4 mol/l). Since the Km of Streptomyces, P. fluorescens, and Cellulomonas enzymes were too low, cholesterol linearity obtained from each source was low. In order to increase the enzyme's Km, we studied the effect of six dichlorophenol isomers. Only 3,4-dichlorophenol significantly raised the apparent Km value of Streptomyces and extended linearity. The high Km of Brevibacterium resulted in an insensitive reaction and low cholesterol linearity. An increase in sample to reagent ratio from 1:100 to 1:10 enhanced the reaction rate and the linearity.
We suggest that Brevibacterium in a sample to reagent ratio of 1:10 and Streptomyces (with the addition of 3,4-dichlorophenol) in a sample to reagent ratio of 1:100 may be considered as appropriate sources of cholesterol oxidase useful for the kinetic cholesterol assay in human serum, however, one must evaluate these enzymes using human serum. For clinical use, we evaluated and compared the performance characteristics of Brevibacterium and Streptomyces sources by the kinetic method for total cholesterol determination in human serum.
Section snippets
Equipment
We used the Vitalab Selectra (E. Merck Dramstadt, Germany) to determine the reaction rate of cholesterol oxidase and to detect cholesterol concentrations in human serum by the kinetic method.
Reagents
We prepared the reagent base by dissolving peroxidase 10 000 U/l, (Sigma Chemical Co., St. Louis MO), sodium cholate, 3 mmol/l (Sigma), 4-aminophenazone, 0.5 mmol/l (BDH Dorset BH 12 4NN, England), phenol 20 mmol/l (Sigma), and Brij 35, 4.5 g/l (Sigma) in phosphate buffer, 0.1 mol/l, pH 7.0.
To prepare the
Optimizing of cholesterol oxidase
To eliminate the effect of cholesterol esterase activity for hydrolysis cholesteryl esters to free cholesterol, we performed the optimization of two cholesterol oxidases with free cholesterol standard in ethanol solution. Fig. 1 shows the optimization of Brevibacterium (A) and Streptomyces enzymes (B) used in the kinetic cholesterol assay. The upper end of the reportable range decreased as the Brevibacterium cholesterol oxidase activity increased. At low enzyme activities (100–500 U/l), we
Discussion
The kinetic method may offer advantages over the endpoint assay, such as shorter analysis time, minimization of interfering substances and no requirement for a sample blank [2], [3]. In our previous study [13], Brevibacterium, in a sample to reagent ratio of 1:10, and Streptomyces (with the addition of 3,4-dichlorophenol) in a sample to reagent ratio of 1:100 provided a suitable reportable range. They may be considered as appropriate sources of cholesterol oxidase for the kinetic cholesterol
Conclusion
Our results suggest that cholesterol oxidase from Streptomyces, in the presence of 3,4-dichlorophenol, is a suitable source of cholesterol oxidase for serum cholesterol assay by the enzymatic kinetic method. Analytical performance of the assay was precise and accurate. For Brevibacterium cholesterol oxidase, a large serum sample was required and the linearity of cholesterol was too low (2.6 mmol/l) for routine clinical work. Therefore, the Brevibacterium enzyme did not appear to be appropriate
Acknowledgement
This study was supported in part by the Thai Research Fund Royal Golden Jubilee PhD program.
References (22)
Enzymatic methods for quantification of lipoprotein lipids
- et al.
Application of cholesterol oxidase in the analysis of steroids
J Chromatogr
(1974) Third report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults
(2001)- et al.
Reaction rate methods of clinical analyses
- et al.
The centrifuged analyzer and its use in clinical analyses
- et al.
Kinetic enzymic method for automated determination of total cholesterol in serum
Clin Chem
(1983) - et al.
Enzymatic determination of total serum cholesterol
Clin Chem
(1974) - et al.
Streptomyces: A superior sources of cholesterol oxidase used in serum cholesterol assay
J Clin Lab Anal
(1992) Preparation and properties of cholesterol oxidase from Nocardia sp. and its application to the enzymic assay of total cholesterol in serum
Clin Chem
(1973)- et al.
Enzymatic assay of total cholesterol involving chemical or enzymic hydrolysis—A comparison of methods
Clin Chem
(1979)
Cholesterol oxidase from Nocardia erythropolis
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