pyridoxal-isonicotinoyl-hydrazone and ferric-nitrilotriacetate

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Topics: Ascorbic Acid; Chelating Agents; Deoxyribose; Edetic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Hydroxyl Radical; Isoniazid; Nitrilotriacetic Acid; Oxidation-Reduction; Oxidative Stress; Pyridoxal

The ability of lymphocytes to utilise iron from different sources has been investigated. Iron uptake from transferrin by proliferating lymphocytes gradually increased as saturation of the protein with iron was increased up to 100%, but rose sharply when addition of further iron resulted in the presence of non-transferrin bound iron. Increasing the saturation of transferrin with iron caused an increased rate of proliferation up to about 100% saturation but when the level of iron present exceeded the binding capacity of the protein, proliferation decreased and at high levels of iron it was reduced below that seen in the absence of transferrin. Comparison of the degree of iron uptake from transferrin and from iron chelators showed that the hydrophilic chelator ferric nitrilotriacetate (FeNTA) donated larger amounts of iron to cells than did transferrin or the lipophilic chelator ferric-pyridoxal isonicotinoyl hydrazone (FePIH), but did not promote proliferation, and when present in high amounts caused inhibition. In contrast, FePIH supported proliferation as efficiently as transferrin. In cells cultured with FeNTA, iron was found predominantly in an insoluble form while in the cells cultured with Fe-transferrin or FePIH the largest proportion of iron was found in the non-ferritin high molecular weight fraction, which probably represents iron in enzymes and other metabolically-important proteins. In no case did iron associated with ferritin exceed 15% of the total uptake, and the cells showed no marked increase in synthesis of ferritin in response to any of the forms of iron. These results indicate that different forms of iron are handled in different ways by lymphocytes, and that iron delivered from hydrophilic chelates may be toxic and not readily available for metabolic use. Lymphocytes appear to be poorly equipped to sequester excess iron in ferritin, and this may account for abnormalities in the immune system reported in patients with iron overload.

Topics: Animals; Cells, Cultured; Chelating Agents; Ferric Compounds; Intracellular Fluid; Iron; Iron Chelating Agents; Isoniazid; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred BALB C; Nitrilotriacetic Acid; Pyridoxal; Transferrin