Sialic acid determination method

Claim 1

Method for determining sialic acid in blood plasma or serum, consisting essentially of the steps of: transferring approximately 50 μl, precisely measured, of serum to a container with 150 μl distilled water; vigorously mixing the container; cooling the container and contents to about 32° F. (0° C.); adding to the container 3 ml of cold (4°-5° C.) 2:1 v/v mixture of chloroform and methanol, and mixing vigorously; adding 0.5 ml cold distilled water to the container and mixing gently; separating the phases in the container by centrifuging; adding 50 μl phosphotungstic acid solution (1 gram per ml) to 1 ml of the upper layer from the centrifuge mixture, and mixing; allowing the phosphotungstic acid solution mixture to stand at room temperature for about 5 minutes; centrifuging the phosphotungstic acid mixture for 5 mins. at 2500 rpm and removing the supernatant; adding 1 ml water to the precipitate from which the supernatant has been removed, and mixing well until precipitate is in suspension without gross particles; adding 1 ml of resorcinol reagent, mixing and placing the container in boiling water for a precise period of time of about 15 minutes; thereafter transfering the container to an ice and water bath and allowing the contents to cool for about 10 minutes; to the cold container contents adding 2 ml butyl acetate-n-butanol 85:15 v/v mixture at room temperature, mixing well and centrifuging for 5 minutes at 2500 rpm; and reading the extractive blue color at 580 nm and determining the amount of lipid bound sialic acid by the use of standard curves developed from a standard sample of n-acetyl neuraminic acid (NANA) under the same conditions and by applying the following formula: LSA mg/100 ml plasma=(X·100,000λ)/(y·44.7λ·1000 x=γ NANA read from standard curve for the sample y=1 ml of supernatant÷volume of entire supernatant (In our experience this had been 1.00/1.30).