An enzyme thermistor-based assay for total and free cholesterol

A method to evaluate the free (FC) and total cholesterol (TC) in human seru m, bile and gallstone extract using an enzyme thermistor (ET)-based flow in jection analysis (FIA) is presented. The cholesterol in high-density (HDL-C ) and low density lipoprotein (LDL-G) have also been evaluated. A heparin f unctionalized Sepharose column was employed for the isolation of HDL and LD L fractions from serum. The estimation of cholesterol and its esters was ba sed on their reaction with cholesterol oxidase (GO), cholesterol esterase ( GE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only GE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutara ldehyde. Column A was used for estimating FC and column C was used for esti mating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunc tion with column A for the estimation of TG or FC, respectively. A calibrat ion between 1.0 and 8.0 mmol/l for FC and 0.25 and 4.0 mmol/l for TC was ob tained. For more than 2000 assays with the ET device a CN. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholest erol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit. (C) 1999 Elsevier Scien ce B.V. All rights reserved.