Four categories of methods are presently used for the quantitative determination of the albumin content of serum or plasma in human blood. These are: (a) Electrophoresis; (b) immunological precipitation with a specific antiserum; (c) spectrophotometric measurement of the color reaction between albumin and hematin; and (d) fractional precipitation with salts or alcohol-buffer mixtures, followed by digestion and chemical colorimetry.The electrophoretic technique of Tiselius has contributed much to knowledge of plasma albumin in biologic systems and is fully discussed in recent review articles by Stern and Reiner ( 1 ), Luetscher (2) and Gutman (3). However, electrophoresis has the disadvantages of being cumbersome and slow, requiring relatively large samples, expensive equipment, and highly skilled technicians. It The hematin method (7) has certain limitations which tend to make it unsatisfactory for general laboratory use. The range of concentration is small and the slope of the calibration curve is such that a relatively large increment in protein concentration is necessary for a measurable change in optical density. The authors (7) stated: "Because of the narrow spectral band width involved in the determination, the photometric measuring instrument must have better resolution than the usual photoelectric colorimeter." They also pointed out, "The possibility of accessory binding by alpha globulin must be borne in mind."The usual clinical laboratory methods for the determination of albumin in serum or plasma depend upon preliminary protein fractionation of the serum, using 21.5 per cent sodium sulfate in the Howe method (8) and 23 per cent sodium sulfate in the Kingsley modification of the Howe method (9), followed by digestion and chemical colorimetry. These salting out methods are not accurate, since they have been shown to give chemical values for albumin 10 per cent to 15 per cent higher than corresponding electrophoretic measurements (10), probably because some of the alpha globulins remain in solution (11-13). The more recent low temperature methods employing methanol-water mixtures with acetate buffers ( 14) or ethanol-water mixtures (13) Method: The optical density of solutions of the dye (HBABA) against a distilled water blank at pH values of 4.1, 5.0, 5.9, 6.2, 6.4, and 7.2 were measured on the Beckman spectrophotometer4 at various wave lengths, and a spectrophotometric absorption curve constructed. These solutions were prepared by adding to 2.5 ml. of the stock dye solution (1 X 10-M) enough acetate buffer at pH 5.0 and 6.2 to reach the desired pH, as measured in a Beckman pH meter. The resulting solutions were each diluted up to 50 ml. with distilled water, and the pH again measured. The final concentration of the dye in the test solutions was 5 X 10'M. This is the final concentration of the dye in all of the spectrophotometric experiments herein reported.Results: At pH values of 5.0, 5.9, 6.2 and 6.4, the curves are nearly identical, the wave length of maximum absorption being 350 m,u in each cas...
