Enzymatic determination of high density lipoprotein phospholipid using a sensitive reagent and a Cobas Bio Centrifugal Analyzer

Abstract

A sensitive enzymatic method for the measurement of high density lipoprotein choline-containing phospholipids (HDL-pc) was adapted to a Cobas Bio Centrifugal Analyzer. The Boehringer Mannheim phospholipase D/choline oxidase/peroxidase reagent was modified by the inclusion of 2,4,6, tribromo-3-hydroxybenzoic acid (TBHBA) which reacts with hydrogen peroxide and 4-aminophenazone to produce a quinoneimine dye with a fourfold greater molar absorption than that produced with phenol. The method has been developed for the determination of HDL fractions isolated with polyethylene glycol 6000, for which a reagent of high sensitivity is required. The method is linear to 5.5 mmol/l of HDL-pc and the CVs for within-run and day-to-day imprecision were less than 3.5%. Correlation of HDL cholesterol (HDL-c) with HDL-pc values in 200 healthy subjects was good (r=0.9024). The mean (±SD) HDL-pc value for men was 1.12 (±0.26) and for women, 1.34 (±0.29) mmol/l. The assay is inexpensive and large numbers of specimens can be processed rapidly and conveniently.

Key Words:

• centrifugal analyzer
• choline oxidase
• Cobas Bio
• end-point enzymatic method
• high density lipoprotein choline-containing phospholipids
• phospholipase D
• polyethylene glycol 6000
• 2,4,6-tribromo-3-hydroxybenzoic acid