A micro enzymic method for determination of choline-containing phospholipids in serum and high density lipoproteins

A micro method is described for the assay of choline-containing phospholipids in serum and high density lipoproteins (HDL) using an automated microtiter plate reader. The method is adapted from the enzymic method of Takayama, Itoh, Nagasaki, and Tanimuzu (Clin. Chim. Acta 79, 93–98, 1977) using phospholipase D, choline oxidase, and peroxidase coupled with the color generating system phenol and 4-amino-antipyrine. The micro method requires 5 μL of serum or HDL sample, and 42 samples can be assayed in duplicate in one run using a 96-well flat-bottom microtiter plate. The reaction is linear up to 400 mg/dL and the lower limit of detection is 0.25 mg of choline-containing phospholipids per assay. The coefficient of variation within an assay is 0.86–0.79%, and day-to-day variation is 0.9–1.5%. Results obtained by the micro method are in excellent agreement with those obtained by the procedure of Takayamaet al. (r=0.997). The supernatant left after removal of low density lipoproteins and very low density lipoproteins from serum and precipitation with heparin/manganese chloride reagent can thus be conveniently used for the micro assay of choline phospholipids in HDL.