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.