Effects of serum storage on the determination of cholesterol

Cholesterol determined by 4 different enzymatic commercial kits and by the dry chemistry Reflotron system was higher in serum stored at 4 °C and at -20 °C than in fresh serum. The effects of storage seem to be temperature-dependent. In fact, cholesterol values significantly increased only after 2h of freezing. The prolongation of freezing up to 2 weeks was not followed by further significant changes. In serum stored at 4 °C the increase in cholesterol was slower than in frozen serum. Both free and esterified cholesterol underwent an increase after storage. When cholesterol was determined by a chemical method (sulfuric acid-ferric chloride) after extraction with ethyl acetate and ethanol, no difference was observed in fresh and stored serum. Cholesterol, triglycerides and apoproteins A-I and B underwent parallel changes after storage both in whole serum and fractionated lipoproteins. Our findings strongly suggest that in serum stored at positive or negative temperature there is an alteration of the lipoprotein molecules which allows an easier availability of cholesterol for the enzyme-substrate reaction than in fresh serum. Current enzymatic methods underestimate (about 10%) cholesterol when the analysis is performed on fresh serum.