Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods

Abstract

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl 2 was assessed and compared with the conventional method using the FeCl 3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl 2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl 3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl 3, due to Mn 2+ ions present in the supernatant. Addition of Mn 2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl 3 reagents showed that Mn 2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl 3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl 2 supernatant was treated with FeCl 3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn 2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.