A simple, rapid, and sensitive method for the determination of iron in serum or plasma is described.The procedure is carried out at room temperature with 2 ml. of serum or plasma, or with 1 ml. if high values are expected; it can be applied to turbid or jaundiced samples, whether previously frozen or not.An ethanolic solution of 4: 7-diphenyl-1: 10-phenanthroline is used to produce a coloured iron complex, the optical density of which can be measured in any suitable photometer, using either 10 or 20 mm. fused glass cuvettes or matched tubes of 1.1 cm. internal diameter.
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...
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