Procedures for Successive Microcolorimetry of Fe²⁺ and Fe³⁺ Bound to Human Serum Protein

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Fe²⁺ and Fe³⁺ bound to protein in human serum were released by addition of TCA-HCl mixture after elimination of coexisting ascorbic acid, uric acid, etc. by centrifugation with an ultrafiltration membrane cone filter or after decomposition of these reducing agents with ascorbate oxidase and uricase. Fe²⁺ in the supernatant of the TCA-HCl mixture was determined at 535nm by the bathophenanthroline method, and the Fe³⁺ in the supernatant was reduced to Fe²⁺ by addition of ascorbic acid and successively assayed as Fe²⁺ by the bathophenanthroline method. Changes in the amount of Fe²⁺ and Fe³⁺ bound to human serum protein over a 24-h period were then studied by using the present method. A peak of Fe³⁺ in human serum seemed to appear in the morning, while almost no peak was observed for Fe²⁺ (average concentration: 10μM). So, the ratio of Fe²⁺/Fe³⁺ bound to human serum protein varied over the range of 0.5-5.0 within a day. The normal range of total iron concentration in human serum was 12-28μM.