Estimation of Hemoglobin on a Basis of Protein Iron

Abstract

There have been several micro-methods in the recent literature for the determination of total iron in blood, 1 , 2 , 3 and it has been suggested that these determinations be used for the calculation of hemoglobin. On account of the constant breakdown of hemoglobin to bilirubin there is a variable quantity of liberated inorganic iron which may amount in certain anemias to more than 5% of the hemoglobin iron. 4 Consequently it would seem advisable to make a preliminary separation of inorganic iron from hemoglobin. Riecker 4 has observed that such a separation may be accomplished by means of a trichloracetic acid precipitation.

In the present study hemoglobin has been calculated from the iron determined in the trichloracetic precipitate from whole blood according to the following technique: A 1 cc. sample of blood is transferred to a 50 cc. centrifuge tube, baked with N/10 HCl and precipitated with trichloracetic acid. After centrifugation the supernatant fluid is poured off, the precipitate dissolved in a minimal quantity of NaOH, transferred to an Erlenmeyer flask, and digested with 5 cc. of nitric acid and 0.5 cc. of 60% perchloric acid. The digest is dissolved in 5 N H₂SO₄, transferred to a 50 cc. volumetric flask and the prussian blue color developed, using gum ghatti as a protective colloid. A standard of known iron content for comparison is prepared at the same time. This technique for determination of protein iron has been used to standardize a dry hemoglobin preparation which is used for the determination of hemoglobin by means of the acid hematin color, somewhat according to the plan of Elvehjem 5 and Arnow. 6

The hemoglobin powder is prepared from blood by precipitating the hemoglobin and other proteins with NaCl at saturation after acidification with hydrochloric acid.