Plasma Protein Distribution by the Modified Wu Colorimetric Method During Glucose Tolerance Tests.

Abstract

Wu 1 published a colorimetric method for the analysis of plasma proteins by use of a phospho-18-molybdictungstic acid (phenol) reagent. The method of fractionation and the standard used was modified by Gibson 2 and his method in essential details has been followed in this work. Six cc. of blood were removed by vena puncture, oxalated and centrifuged to obtain the clear plasma. One cc. of the plasma thus obtained was placed in a 15 cc. centrifuge tube and 2 cc. of distilled water added. To this was added 1 cc. of saturated ammonium sulphate thus bringing the solution to one-fourth saturation, precipitating out the fibrinogen. After standing 10 minutes the fibrinogen was lightly centrifuged and the fluid decanted into a dry tube. One cc. of the fluid was placed into a second 15 cc. centrifuge tube with 1/2 cc. of water and 1 cc. of saturated ammonium sulphate. This brought the mixture to half saturation thus precipitating out the globulins. After standing 15-20 min. the tubes were centrifuged rapidly, and the supernatant fluid decanted off. One cc. of this fluid and 2 cc. of water were pipetted into a third centrifuge tube for the albumin determination.

After all the liquid had been drained from the fibrinogen and globulin, the precipitates were dissolved in 3 cc. of water. From this point the separated fractions were treated in the same manner. To each tube was added 1 cc. of trichloracetic acid (20%) and the resulting precipitate centrifuged out. The fluid was removed, 2 cc. of distilled water blown from a pipette so as to cause a dispersion of protein and one drop of concentrated sodium hydroxide added to cause solution. The volume was brought to 9 cc.