Improved methods for the quantitative determination of plasma proteins

Abstract

The blood is drawn into a tube containing an amount of potassium oxalate sufficient to make 0.2 or 0.3 per cent. oxalate solution, and is centrifuged twenty minutes.

Fibrin.—5 c.c. of plasma are run into a beaker containing 100-150 c.c. 0.8 per cent. NaCl and 2-5 c.c. of a 2.5 per cent. CaCl₂ solution. The CaCl2 may be in amounts from 2-25 equivalents of the oxalate, but about five equivalents are best. When coagulation is complete, the fibrin is filtered, the clot washed with 0.8 per cent. NaCl, and the nitrogen determined by Kjeldahl. The above is an adaptation of Howell's method for determining the activity of thrombin. 1 The filtrate from the clot may be tested for complete precipitation by addition of a solution containing thromboplastic substances.

Albumin and Globulin are calculated from the following three determinations:

Total nitrogen is determined on a 1-2 c.c. sample.

Non-protein nitrogen is determined in the filtrate obtained after precipitating the plasma with nine volumes of trichloracetic acid.

Nitrogen of Globulin Filtrate:—Globulin and fibrin are precipitated by adding to 5 c.c. of plasma 20 c.c. of H₂O and 25 c.c. of saturated ammonium sulfate solution. 20 c.c. of the filtrate are mixed in a Kjeldahl flask with 3 gm. MgO “Merck's Reagent” and 350 c.c. of 50 per cent. alcohol. The solution is distilled until the distillate gives a negative test to red litmus. This takes about one hour and reduces the volume to about 20 c.c. The nitrogen, representing albumin plus non-protein nitrogen, is then determined by Kjeldahl, using 25 c.c. H₂SO₄. When the digestion mass becomes light brown, the sides of the flask are washed down with a few c.c. of water and ten more c.c. H₂SO₄ added.