Determination of the serum iron-binding capacity

Abstract

SYNOPSIS A colorimetric and an isotopic method for determining the iron-binding capacity are described, using only 1 ml. samples of plasma or serum. Both methods are simple, rapid and sensitive. Satisfactory results are obtained from frozen as well as from fresh samples.A simple colorimetric method for estimating the iron content of plasma, using an ethanolic solution of bathophenanthroline to form a coloured iron complex under controlled conditions, has been described by Kok and Wild (1960).The procedure, which gives accurate results, has been employed in measuring the total iron-binding capacity of plasma or serum after the iron-binding protein has been saturated by the addition in vitro of an excess of ammonium ferric citrate and the unbound excess has been removed by a basic anion exchange resin. This technique was used first by Peters, Giovanniello, Apt, and Ross (1956), whose findings are confirmed by the data recorded in this paper.An isotopic method of measuring the total ironbinding capacity, also based on the technique of Peters et al. (1956), has been described by Bothwell, Jacobs, and Kamener (1959). A modification of their method, using the same reagents necessary for the colorimetric procedure described in this paper, is shown to give satisfactory results. REAGENTS IRON-FREE WATER Water, distilled twice over glass, was passed through an Elgastat B102 deionizer. The resistance of this water was greater than 6 x 106 ohms/cm. AMMONIUM FERRIC CITRATE AnalaR ammonium ferric sulphate, 0-430 g., was dissolved in water and ferric hydroxide precipitated by dilute ammonia. After centrifuging the precipitate was dissolved on warming with a few crystals of citric acid. The pH of the solution was adjusted to 6 5 to 71 by the addition of dilute ammonium hydroxide and the solution diluted to 1 litre.The solution, containing 50 ,ug. Fe/mi., was stored at 40C.Ammonium ferric-59Fe citrate (A) for the determination of the unsaturated iron-binding capacity was preparedReceived for publication 23 December 1964. by adding 0-1 ml. 59ferric chloride (5 XC., 1 ,zg. Fe) to 5 ml. of the above solution of ammonium ferric citrate.BUFFER pH 7-5 Sodium chloride, 6-4 g., and 2-3 g. sodium salt of diethyl barbituric acid were dissolved in 1 litre of iron-free water. Then 6-0 g. diethyl barbituric acid was dissolved in the solution. The pH of the buffer was checked1 and found to be 7-5.AMBERLITE IRA 410 RESIN The resin was suspended in 4N hydrochloric acid for 48 hours to remove any iron and saturate the resin with chloride ions. It was washed with iron-free water, suspended in the pH 7 5 buffer, and dried at 95°C. The reagents for the serum iron determination were made up as in the method of Kok and Wild (1960). APPARATUSAll the glass used was washed in detergent solution, soaked in 5000 hydrochloric acid for 48 hours, and then rinsed three times with iron-free water. PROCEDURE 1 COLORIMETRIC METHOD (a) Serum or heparinized plasma, 1 ml., is pipetted into a 10 ml. graduated conical centrifuge tube and 0-10 ml. of ammonium ferri...

Method for the determination of unsaturated iron-binding capacity of serum using radioactive iron and magnesium carbonate

“…This is in agreement with reports in the literature that there is practically no difference in UIBC measured by a radioactive iron method using ion exchange resin and by a colorimetric or immunodiffusion technique (Birdsall et al, 1965;Charlton et al, 1965;Hillman, Morgan, and Finch, 1967;Stojceski et al, 1965;Tauxe, 1961). On the other hand, Burrows (1967), Herbert et al (1966), and Koepke (1965) found that UIBC values measured by the radioactive iron method with a resin were approximately 100 ,ug Fe/100 ml higher than measured by other methods.…”

“…The presence of particles in the supernatant considerably increases the optical density of a sample and affects the colorimetric measurement. A number of investigators have observed that the use of the methods involving ion exchange resin give falsely high results for UIBC (Charlton, Hardie, and Bothwell, 1965;Stojceski, Malpas, and Witts, 1965;Birdsall, Kok, and Wild, 1965). Charlton et al (1965) suggested that the absorption of water by the dry resin, with a proportional increase in the concentration of all substances in the supernatant, results in increased UIBC, and that this can be overcome by counting the radioactivity in the supernatant.…”

“…However, this means washing the resin which prolongs the procedure. The other modifications of the original method of Bothwell et al (1959) by Stojceski et al (1965 and Birdsall et al (1965) proposed a change in the order of adding the reagents. These workers claim to have obtained good results in a comparison with the colorimetric method.…”

Intravenous iron-dextran: studies on unsaturated iron-binding capacity

“…Plasma unsaturated iron-binding capacity determinations were made by a modification of the method of Birdsall, Kok, and Wild (1965). The general procedure was as published in that paper, but it was found necessary to remove the excess unbound 59Fe ammonium citrate which was added to the plasma sample by absorption on a small column (5 x 1 cm) of Amberlite IRA-400 resin.…”