A Method for the Estimation of Serum Iron. ∗

Abstract

The drawing of blood under conditions aiming to avoid hemolysis, as described by Fowweather, 1 has yielded in our hands serum that invariably gives a positive benzidine test and shows the absorption bands of oxyhemoglobin if a sufficient thickness of solution is examined. The present method for the estimation of the non-hemoglobin iron of blood serum consists of 2 steps, the analysis of the serum for total iron and for hemoglobin iron.

The total iron is determined by ashing 2 cc. of serum with 2 cc. of concentrated sulfuric acid, with the aid of 30% H₂O₂. The ashed sample is diluted to 15 cc. with water, enough potassium permanganate is added to give a permanent pink color, 5 cc. of ethyl acetate are layered over the solution and, finally, 5 cc. of 20% ammonium thiocyanate solution are added and the mixture is shaken. The color in the ethyl acetate layer is compared in a micro-colorimeter with a standard containing 0.005 mg. of iron similarly treated. The traces of iron in the reagents are determined by blank analyses.

The quantitative benzidine method 2 is employed for the determination of the hemoglobin, but allowance must be made for the effect of serum proteins on the reaction. Proteins and certain salts cause a diminution in the color produced in the benzidine acetate-hemoglobin-H₂O₂ system. The method may be summarized briefly. To 2 cc. of the benzidine reagent 0.5 cc. of blood serum and 0.5 cc. of water are added, followed by 1 cc. of 0.6% H₂O₂ solution. In another test tube the same procedure is carried out, except that 0.5 cc. of the standard solution of blood, containing 0.05 mg. of hemoglobin per cc., is used in place of 0.5 cc. of water.