A Multicommuted Flow Procedure for the Determination of Cholesterol in Animal Blood Serum by Chemiluminescence

Abstract

An automatic flow procedure for cholesterol determination in animal blood serum using enzymatic reaction and detection by chemiluminescence is described. Cholesterol esterase and cholesterol oxidase were immobilized on glass beads packed into two mini-columns (15 mm × 5 mm), which were coupled to flow system. Produced H₂O₂ reacted with luminol produced chemiluminescence that was detected using a solid-state detector. The flow system comprised a set of three-way solenoid valves assembled to implement multicommutation. A microcomputer with an electronic interface controlled the solenoid valves and performed data acquisition. Profitable features such as a sampling throughput of 40 determinations per hour, linear response ranging from 25 to 125 mg L⁻¹ cholesterol (r = 0.998), detection limit of 3.7 mg L⁻¹ cholesterol, relative standard deviation of 2.3% (n = 20), and low reagents consumption 0.22 mg luminol and 2.7 mg hexacyanoferrate(III) per determination were achieved. Comparing the results with those obtained using the conventional spectrophotometric method no significant difference at the 95% confidence level was observed.

Keywords:

• Flow analysis
• Multicommutation
• Chemiluminescence
• Cholesterol
• Animal blood
• Enzymatic reaction

Acknowledgments

Authors are gratefully to the Laboratory of Animal Nutrition (CENA/USP) for providing the blood samples, CAPES, CNPq/PRONEX, and FAPESP for the financial support.