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Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.

We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxal isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20-200 microM 59Fe-PIH. The amount of 59Fe incorporated with 200 microM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 microM 59Fe concentration. After 60 min about 60% of the 59Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of 59Fe into heme by about 70%, indicating that 59Fe from 59Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate 59Fe from 59Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate 59Fe from 59Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.

A model consisting of 59 Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59 Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59 Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides perpared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59 Fe release from 59 Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59 Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrzaones. Several compounds proved to be very potent in mobilizing 59 Fe. These included hydrazones derived from 2-hydroxyl-1-naphthylaldehyde and benzoic acid hydrazide, p -hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m -fluorobenzyol hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.

Cobalt was found to stimulate globin synthesis in reticulocytes the heme synthesis of which was inhibited by isonicotinic acid hydrazide or 4,6-dioxoheptanoic acid (succinyl acetone). These results suggest that in reticulocytes cobalt does not stimulate globin synthesis through the formation of cobalt protoporphyrin. They do, how-ver, support the hypothesis that cobalt causes the formation of a pool of free heme from preformed hemoglobin which stimulates the synthesis of new globin chains.

An in vitro system is described in which iron incorporation into rabbit reticulocytes and bone marrow cells from pure transferrin is qualitatively comparable to that found in vivo. The amount of stromal-bound iron reproducibly represents 2% and 12% of the total iron incorporated respectively into reticulocytes and bone marrow cells after 2.5 h of incubation. After 60 min of incubation only 40% of the reticulocyte stromal-bound iron behaved like a metabolic intermediate. All the metabolically active, membrane-bound iron could be accounted for a transferrin iron. Of the transferrin found in washed cells, 50–60% was recovered in the cytoplasmic fraciton, and was shown by isoelectric focusing to be undergraded. It is likely that it represents intracellular transferrin. The rate of hemoglobin synthesis was linear for at least 2.5 h in both reticulocytes and bone marrow cells, while bone marrow cells showed z greater iron incorporation into heme than reticulocytes. There was no detectabel low molecular weight iron in the cell supernatant of reticulocytes or bone marrow cells and hemoglobin-iron accounted for more than 92% of the total iron incorporated into the reticulocytes.

The soluble, labeled product of a cell-free system derived from rabbit reticulocytes was investigated to determine its role in the assembly of hemoglobin (Hb). It has been concluded that the cell-free system synthesizes a protein which by two independent criteria is not yet a finished Hb molecule. The product was found to be more basic than Hb on ion-exchange chromatography and to have a molecular weight, as determined by gel filtration and sucrose density centrifugation, characteristic of a dimer. The dimer is unstable but can be converted, in part, to a stable Hb-like molecule by exogenous hemin. These findings suggest that the dimer is an intermediate in the synthesis of Hb. The significance of this is discussed in relation to the regulation of Hb synthesis.

A method is described for the preparation of concentrated suspensions of viable rabbit reticulocytes from animals rendered anemic by repeated bleeding. The method involves the centrifugation of washed red blood cells through dextran density gradients.

By separating breis derived from soybean plant root nodules into soluble and particulate components and incubating each with δ-amino[4−14C]levulinic acid, it was found tha: (I) The heme synthesized by the nodules is principally the product of the particulate component. (2) The particulate component is predominantly bacteriods and is devoid of plant mitochondria. (3) Some of the heme synthesized in the breis is incorporated into leghemoglobin. We conclude that bacteroids in the nodules are responsible for the major part of the synthesis of heme for leghemoglobin.