Method for determining lipid bound sialic acid in plasma

A method of extracting lipid bound sialic acid from human blood plasma or serum and determining the amount of lipid bound sialic acid in a sample of human blood plasma or serum which comprises the following steps:

• a) diluting a predetermined volume of 25 microliters or less of a blood plasma or serum sample with distilled water to a volume about two times that of the predetermined volume of the sample;
• b) mixing the diluted sample for a suitable period of time to obtain a substantially homogeneous sample;
• c) adding to the sample a mixture of a chlorinated lower alkyl hydrocarbon and a lower alkyl alcohol, the volume of the mixture added being about forth times the predetermined volume of the blood plasma or serum sample, and the volume ratio of chlorinated hydrocarbon to alcohol in the mixture being about 2:1;
• d) mixing the resulting admixture for a suitable period of time to dissolve lipid-bound sialic acid in the sample in the chlorinated hydrocarbon/alcohol mixture;
• e) diluting the admixture with a buffer solution at room temperature, the volume of buffer solution being about eight times the predetermined volume of the blood plasma or serum sample;
• f) mixing the diluted admixture without vortexing for a suitable period of time to obtain a substantially homogeneous admixture and centrifuging the mixture to form a substantially clear upper phase;
• g) separately recovering from the clear upper phase so formed a predetermined volume of the upper phase;
• h) adding to the predetermined volume of the upper phase an amount of a mixture of a protein-precipitating agent and water without any adsorbing material, the amount of mixture being effective to cause precipitation of the lipid bound sialic acid;
• i) mixing the resulting admixture;
• j) separately recovering the resulting precipitate;
• k) suspending the precipitate in a hydrolysis agent;
• l) determining the amount of lipid bound sialic acid present in the suspended precipitate and thereby the amount present in the blood plasma or serum sample.