Distribution of Intravenously Administered Ferrioxamine-59 and Ferric N, N'-Ethylenebis (a Imino-2-Hydroxy-5-Chlorophenylacetate) in Rats

Abstract

The relative importance of molecular structure in comparison to quantitative iron binding capability of iron chelates has not been delineated for the problems of the membrane transport of iron, the distribution of the metal in vivo and its excretion (1,2). Ferrioxamine and ferric N,.V' ethylenebis-( a imino-2 -hydroxy-5-chlorophenylacetate) (FeCI-EDDHA) are two iron chelates of differing molecular architecture whose iron binding strength exceeds that of iron transferrin (3, 4 ) . The present study suggests that the molecular environment of the metal is a major determinant in its biological fate.Materials and Methods. Preparation of tagged chelates. Ferrioxamine and FeCI-E D D H A were prepared by metathesis from ferric chloride and a 20% molar excess of the ligand dissolved in 0.2 M sodium carbonate solution. Radioactive ferric chloride was added (0.1 pC/ml) and the p H was adjusted to 7.3 with sodium carbonate. The iron concentration was 2.8 mg/ml. The solutions so prepared were intensely colored and stable to hydrolysis.In vitro transfer of iron to P-globulin. Mixtures of plasma and chelate, 0.30, 0.15, 0.75, and 0.030 m M chelate in plasma, were incubated at 37°C for 15 min. These solutions were the equivalent of 100, 50, 25, and 10% of the dosage administered to a rat and calculated as wholly diluted in the plasma. After incubation!, the mixtures were subjected to electrophoresis in barbital buffer on cellulose acetate strips (5). The unstained strips were scanned for radio iron in a 4~ detector' and a duplicate strip was stained for visual location of the beta-globulin fraction. Scanner, Model RSC-363.In vivo studies. Fasted, male Sprague-Dawley rats (150-200 gm) were used in these experiments. The animals were iniected in the saphenous vein while under i.p. sodium pentobarbital anesthesia (35 mg/kg). The iron dosage was 6 mg of Fe/kg. At prescribed time intervals each rat was exsanguinated by cardiac puncture, quickly opened 'by an abdominal incision, and the kidneys, liver, gut, spleen, and bladder were removed. Two people working simultaneously could accomplish the entire procedure within 30 sec. Radio iron content was determined in a Baird Atomic well (model 810A). The results were compared to a counting standard representing 1% of the injected dose and the percentage of the dose present calculated for each sample. ThR blood volume was calculated as 6.5% of the body weight. Groups of three animals were used for each time interval studied.Results and Discussion. In vitro transfer of iron to beta-globulin. The radioscan and integration of the strips obtained from this study showed that 15 min after incubation less than 0.5% of the rad'ioactive iron in the iron chelate was transferred to the betaglobulin fraction, as established by the electrophoretic technique. The chdates were clearly discernable as an intense colored band on the unstained strips well in advance of the beta-globulin fraction determined by Ponceau S Shining. For ferrioxamine, these results substantiate findings reported by Keberle (4). Th...