Bilirubin and hemoglobin interferences in direct colorimetric cholesterol reactions using enzyme reagents

The interferences of bilirubin and hemoglobin were tested in two cholesterol procedures in which enzymes were used as chemical reagents. Both procedures used similar approaches with cholesterol esterase to free cholesterol from its esters and cholesterol oxidase to generate hydrogen peroxide from the total free cholesterol resulting. From that common start, one procedure then used catalase to generate formaldehyde from methanol and the peroxide produced from cholesterol, and the formaldehyde was then reacted with acetylacetone to produce a yellow chromogen, while the other procedure used peroxidase to catalyze a reaction directly between peroxide and 4-aminoantipyrine plus phenol to generate a pink chromogen. Bilirubin and hemoglobin were shown to produce some interference by reacting competitively with peroxide in both systems and by contributing residual absorbance at the wavelengths of measurement of each of the chromogens. Since bilirubin showed a spectral change, static blanking with sample blanks caused overcorrections. However, the elimination of a sample blank for either procedure could result in a favorable compensating error because the residual color of bilirubin could substitute in part at least for the lost reactivity of the peroxide used up in reaction with the bilirubin of the sample.