Pyridoxal Benzoyl Hydrazone Research Articles

Effects of changing ions on the crystal design, non-covalent interactions, antimicrobial activity, and molecular docking of Cu(II) complexes with a pyridoxal-hydrazone ligand.

The present work reports the influence of the presence of different ions (Cl-, Br-, NO3 -, or SO4 2-) on the formation and proprieties of Cu(II) complexes with pyridoxal-benzoylhydrazone (PLBHZ). Four new complexes were successfully synthesized, [CuCl2(PLBHZ)] (1), [CuBr2(PLBHZ)] (2), [CuCl(PLBHZ)H2O]⋅NO3⋅H2O (3), and [CuSO4(PLBHZ)H2O]⋅3H2O (4), and characterized by spectroscopic and physicochemical methods. A single-crystal X-ray study reveals the Schiff base coordinated to the metal center tridentate by the ONS-donor system, resulting in distorted square pyramidal coordination geometries. Noncovalent interactions were investigated by 3D Hirshfeld surface analysis by the d norm function, 2D fingerprint plots, and full interaction maps. The ion exchange is important in forming three-dimensional networks with π⋅⋅⋅π stacking interactions and intermolecular hydrogen bonds. The in vitro biological activity of the free ligand and metal complexes was evaluated against Gram-positive and Gram-negative bacterial strains and the free pyridoxal-hydrazone ligand showed higher activity than their Cu(II) complexes. Molecular docking was used to predict the inhibitory activity of the ligand and complexes against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria.

Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe 3+–chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe 3+ complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe 3+ complexes, and toxicity of the chelators increased with oxygen tension. Fe 3+ complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH) 2 did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH) 2 in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe 3+ complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.

Biliary Iron Excretion in Rats Following Treatment With Analogs of Pyridoxal Isonicotinoyl Hydrazone

AbstractIron overload is a major life-threatening complication of thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise for the treatment of iron overload. More recently, over 40 analogs of PIH were synthesized, and some of them proved to be very potent in mobilizing 59Fe in vitro from 59Fe-labeled cells. In this study, we show that PIH analogs such as pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH), pyridoxal m-fluorobenzoyl hydrazone (PFBH), and pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to mobilize iron from cells in vitro, are also effective in vivo. All of these chelators significantly enhanced biliary excretion of iron (measured by atomic absorption spectrophotometry) following their intraperitoneal (IP) and/or oral administration to rats. The most effective was PFBH, which increased iron concentration in the bile about 150-fold, as compared with basal biliary iron concentration, within 1 hour following a single IP dose of 0.2 mmol/kg body weight. In contrast, desferrioxamine increased the biliary iron concentration only 20-fold to 30-fold under the same conditions. Moreover, while control rats excreted ≈ 0.8 μg Fe in 2 hours, treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative excretions of 87, 59, and 22 μg Fe, respectively, in the same period of time. Interestingly, PMBH was also quite effective following gastric administration, resulting in a 6-hour cumulative value of 34 μg Fe. These compounds are nontoxic and are inexpensive and easy to make. Their further evaluation as candidate drugs for the treatment of iron overload is warranted.

Biliary Iron Excretion in Rats Following Treatment With Analogs of Pyridoxal Isonicotinoyl Hydrazone

Iron overload is a major life-threatening complication of thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise for the treatment of iron overload. More recently, over 40 analogs of PIH were synthesized, and some of them proved to be very potent in mobilizing 59Fe in vitro from 59Fe-labeled cells. In this study, we show that PIH analogs such as pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH), pyridoxal m-fluorobenzoyl hydrazone (PFBH), and pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to mobilize iron from cells in vitro, are also effective in vivo. All of these chelators significantly enhanced biliary excretion of iron (measured by atomic absorption spectrophotometry) following their intraperitoneal (IP) and/or oral administration to rats. The most effective was PFBH, which increased iron concentration in the bile about 150-fold, as compared with basal biliary iron concentration, within 1 hour following a single IP dose of 0.2 mmol/kg body weight. In contrast, desferrioxamine increased the biliary iron concentration only 20-fold to 30-fold under the same conditions. Moreover, while control rats excreted ≈ 0.8 μg Fe in 2 hours, treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative excretions of 87, 59, and 22 μg Fe, respectively, in the same period of time. Interestingly, PMBH was also quite effective following gastric administration, resulting in a 6-hour cumulative value of 34 μg Fe. These compounds are nontoxic and are inexpensive and easy to make. Their further evaluation as candidate drugs for the treatment of iron overload is warranted.

Structural analogs of pyridoxal benzoyl hydrazone with improved fibrosuppressive activity in cell culture

Structural analogs of pyridoxal benzoyl hydrazone with improved fibrosuppressive activity in cell culture

Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu).

Artemisinin is an important new antimalarial agent containing a bridged endoperoxide. The in vitro antimalarial activity of an artemisinin derivative, arteether, is antagonized by two iron chelators, pyridoxal benzoylhydrazone and 1,2-dimethyl-3-hydroxypyrid-4-one. Similarly, the acute toxicity of artemisinin in mice is antagonized by another chelator, deferoxamine-hydroxyethylstarch. A combination of artemisinin and hemin oxidizes erythrocyte membrane thiols in vitro, and this oxidation is also inhibited by an iron chelator. Thus, iron plays a role in the mechanisms of action and toxicity of artemisinin. The combination of artemisinin and hemin also decreases erythrocyte deformability. Iron probably catalyzes the generation of free radicals from artemisinin since alpha-tocopherol antagonizes the thiol-oxidizing activity of artemisinin and since a spin-trapped free radical signal can be seen by electron paramagnetic resonance only when artemisinin is incubated in the presence of iron.

Étude Infrarouge du Pyridoxal Isonicotinoyle Hydrazone Et de Composes Analogues

AbstractInfrared spectra have been obtained for pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone, salicylaldehyde isonicotinoyl hydrazone and salicylaldehyde benzoyl hydrazone. Vibrational assignments are proposed mainly in the 2000‐1200 cm−1 region. Evidence is given that a intramolecular bond exists between the phenolic hydroxyl and the nitrogen of imine group. Trans amide III band located near 1550 cm−1 presents a drastic shift from usual results (more than 50 cm−1). Conformational equilibrium is discussed with AM1 and ab‐initio methods. IR and NMR (NOE measurements) studies show that one conformation seems to be predominant.

Synthesis, magnetic and electrochemical properties of binuclear copper(II) complexes of pyridoxal hydrazones

Some new binuclear copper(II) complexes with pyridoxal benzoylhydrazone (PBH), pyridoxal o-hydroxylbenzoyl- hydrazone (PHBH), pyridoxal picolinoylhydrazone (PPH), pyridoxal nicotinoylhydrazone (PNH), and pyridoxal isonicotinoylhydrazone (PINH) have been synthesized and characterized by analytical, magnetic, spectral, ESR and electrochemical methods. All the complexes exhibit normal magnetic moments at room temperature. The variable temperature magnetic moments, however, indicate the presence of very weak intramolecular antiferro- magnetic interactions (−2 J ≈~90 cm −1) in between the copper(II) centers in the complexes. The ESR spectral data at 77 K are characteristic of binuclear CuCu complexes and exhibit a seven-line pattern in the low field g l region. The cyclic voltammograms of each complex showed two oxidation waves attributed to Cu 2 II,II→Cu 2 II,III and Cu 2 II,III→Cu 2 III,III processes at significantly low potentials.

Kinetic and thermodynamic study of complex formation between iron(II) and pyridoxal isonicotinoylhydrazone and other synthetic chelating agents

In neutral aqueous media pyridoxal isonicotinoylhydrazone (PIH), pyridoxal benzoylhydrazone (PBH), salicylaldehyde isonicotinoylhydrazone (SIH), and salicylaldehyde benzoylhydrazone (SBH), which show interesting potential for iron mobilization in living cells, form bicomplexes with Fe(II) in two kinetically distinguishable steps. the first is fast and is ascribed to complex formation with a single ligand

Evaluation of the iron chelation potential of hydrazones of pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the hepatocyte in culture.

A range of new analogues of the promising iron chelator pyridoxal isonicotinoyl hydrazone was prepared and assessed for activity in reducing hepatocyte iron, mechanism of action and potential in iron-chelation therapy. A total of 45 compounds were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring or by the replacement of the ring with an acetyl, pyridyl, furoyl or thiophene moiety. The effects of these compounds on 59Fe uptake and intracellular distribution in hepatocytes in culture and on 59Fe mobilization from prelabeled hepatocytes were assessed. Toxicity, lipophilicity and the ability to chelate plasma transferrin-bound 59Fe were also evaluated. Several compounds were much more active than pyridoxal isonicotinoyl hydrazone and may have clinical potential. These included pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-pyridyl hydrazone. All were more effective at reducing iron uptake than mobilizing hepatocyte iron; they also may act primarily on the transit iron pool rather than on storage iron. Other compounds (e.g., salicylaldehyde p-t-butyl-benzoyl hydrazone) redistributed ferritin-59Fe to different intracellular sites but had little net effect on hepatocyte iron levels.

Mechanism of protection of alveolar type II cells against paraquat-induced cytotoxicity by deferoxamine

Paraquat toxicity has been associated with the generation of free radicals in alveolar epithelial cells in which paraquat specifically accumulates via a polyamine uptake system. In the present study we investigated whether deferoxamine (DF), an iron chelator that has antioxidant capacity and that also has a polyamine-like structure, could protect alveolar type II cells (ATTC) against injury by paraquat. Radiolabeled [ 3H] adenine ATTC were incubated in a medium containing 75 μM paraquat in the absence or presence of DF (500 μM). After 3hr of incubation paraquat-mediated cytotoxicity of ATTC, as measured by [ 3H]adenine release, was significantly (P < 0.005) decreased by addition ofDF (26.6 ± 2.6% vs 7.4 ± 1.7%). Accumulation of radiolabeled [ 14C]paraquat at a concentration of 75 μM was also decreased (70%) by 500 μM DF from 94.8 ± 2.1 to 28.9 ± 6.7 nmoles paraquat / 2.5 × 10 5 ATTC. This effect of DF was dose dependent and comparable with the protective effect of equimolar concentrations of putrescine. However, per cent uptake of paraquat at a concentration of 500 μm was not significantly inhibited by DF (1 mM), whereas paraquat-induced injury was still markedly reduced (36.2 ± 2.5% vs 2.6 ± 4.2%). This indicated that the protective effect of DF could not be explained by its competition with paraquat on uptake alone. In the same series of experiments using another iron chelator, pyridoxal benzoyl hydrazone (PBH), which has antioxidant properties similar to DF but does not show its polyamine-like structure, ATTC lysis was also prevented although paraquat uptake was not reduced. These in vitro data indicate that the mechanism of protection by DF against paraquat toxicity in lung epithelial type II cells is two-fold: inhibition of paraquat uptake through its compliance with the structural requirements necessary for transport, and inhibition of paraquat-induced iron-catalysed free radical generation.

Iron chelators of the pyridoxal isonicotinoyl hydrazone class Part I. Ionisation characteristics of the ligands and their relevance to biological properties

The orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), and five analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone ((PpMBH), pyridoxal m-fluorobenzoyl hydrazone (PmFBH), 3-hydroxy- isonicotinaldehyde isonicotinoyl hydrazone (IIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) were synthesised and characterised and their acid dissociation constants measured by glass electrode potentiometry and UV—Vis spectrophotometry. Analysis of the data showed that at physiological pH all of the ligands are predominantly (av. 80%) in the form of the neutral molecule, allowing passage through cell membranes and access to intracellular iron pools. The results are discussed in the context of the development of an orally effective iron chelator for clinical use.

Iron chelators of the pyridoxal isonicotinoyl hydrazone class Part II. Formation constants with iron(III) and iron(II)

Formation constants for the iron(III) complexes of the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and three analogues: pyridoxal benzoyl hydrazone (PBH), 3-hydroxy- isonicotinaldehyde isonicotinoyl hydrazone (IIH) and salicylaldehyde isonicotinoyl hydrazone (SIH), have been determined by a combination of spectrophotometry and potentiometry. All four ligands bind iron(III) strongly giving, at physiological pH 7.4, values of pM (−log[uncomplexed metal]) between 27.7 and 50, comparable to or greater than those for transferrin (25.6) and desferrioxamine B (28.6). The complexation of Fe(II) by PIH has also been studied and has been found to be appreciable but very much weaker than that for Fe(III).

Pyridoxal isonicotinoyl hydrazone and analogues. Study of their stability in acidic, neutral and basic aqueous solutions by ultraviolet-visible spectrophotometry.

The ultraviolet-visible absorption spectra of the orally effective iron chelator, pyridoxal isonicotinoly hydrazone (PIH), and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone (PpMBH) and pyridoxal m-fluorobenzoyl hydrazone (PmFBH) have been measured in aqueous solution with various concentrations of added acid or alkali. Assignment of absorption bands to various molecular species in equilibrium in aqueous solution is made by reference to their acid ionisation constants. All four hydrazones were stable at physiologial pH, but hydrolysed in strongly acidic and basic solutions, resulting in the liberation of pyridoxal and the acid hydrazide. In acidic solutions this resulted in a dramatic decrease in the intensity of absorption at wavelengths of 225 nm and above 300 nm, allowing a quantitative estimate of the degree of acid-catalysed hydrolysis of the ligands. These results indicate that for oral administration the chelator should be administered with calcium carbonate or provided with an enteric coating to minimise acid-catalysed hydrolysis in the stomach. At high pH, base-catalysed hydrolysis occurred, resulting in a decrease in the absorption at a wavelength of 387 nm.

Iron chelators of the pyridoxal isonicotinoyl hydrazone class

Formation constants for the calcium(II), magnesium(II) and zinc(II) complexes of the orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH) and three analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone (PpMBH) and pyridoxal m-fluorobenzoyl hydrazone (PmFBH) have been determined by potentiometry at 25 degrees C and I = 0.1 M [KNO3]. The four ligands bind calcium(II) weakly and magnesium(II) only slightly more strongly, as a 1:1 complex which is formed at pH greater than 8. The chelation of zinc(II) for all the ligands studied was greater than that for calcium(II) and magnesium(II), with complexation generally becoming significant at about pH 5. Thus, chelation of zinc(II) but not calcium(II) or magnesium(II) at physiological pH, 7.4 may be expected. Calculated values of the concentration of uncomplexed metal ion indicate that the selectivity of these ligands towards Fe(III) is comparable to that of the clinically used chelator desferrioxamine.

Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes

A model consisting of 59Fe-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 59Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59Fe 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 59Fe release from 59Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59Fe. 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 59Fe. 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.

Syntheses of iron(III) aroyl hydrazones containing pyridoxal and salicylaldehyde. The crystal and molecular structure of two iron(III)-pyridoxal isonicotinoyl hydrazone complexes

Iron(III) complexes of three aroyl hydrazones, pyridoxal isonicotinoyl hydrazone (H 2pih), pyridoxal benzoyl hydrazone (H 2pbh), and salicylaldehyde benzoyl hydrazone (H 2sbh), were synthesized and characterized. In aqueous medium at pH 7, [Fe(pih)(Hpih)]·3H 2O is formed. In acidic methanol, a 1:1 ligand-to-metal complex is formed, [FeCl 2(H 2pih)]Cl ( 1), whereas in aqueous medium at low pH cis-[FeCl 2(H 2pih)(H 2O)]Cl·H 2O ( 2) is formed. Compounds 1 and 2 are high-spin d 5 with μ eff = 5.88 μ B and 5.93 μ B (298 K). The crystal structures of 1 and 2 show that H 2pih acts as a tridentate neutral ligand in which the phenolic and hydrazidic protons have shifted to the pyridine nitrogen atoms. The co- ordination polyhedron of 1 is ‘square’ pyramidal, whereas that of 2 is pseudo-octahedral. Compound 1 is triclinic, space group P l , with a = 12.704(2) Å, b = 8.655(2) Å, c = 8.820(2) Å, α = 105.42(1)°, β = 89.87(1)°, γ = 107.60(1)°, V = 888 Å 3, and Z = 2; 2 is monoclinic, space group P2 1/ c, with a = 15.358(4) Å, b = 7.304(3) Å, c = 17.442(4) Å, β = 101.00(2)°, V = 1921 Å 3, and Z = 4.

Iron chelation by pyridoxal isonicotinoyl hydrazone and analogues in hepatocytes in culture

Pyridoxal isonicotinoyl hydrazone (PIH) and several analogues were synthesized and assessed in the rat hepatocyte culture for their potential in iron chelation therapy. Pyridoxal isonicotinoyl hydrazone and pyridoxal benzoyl hydrazone were as effective as desferrioxamine (DFO) in reducing both net uptake of rat transferrin- 59Fe and incorporation into ferritin by hepatocytes. Dialysis studies showed that this was due to a cellular action and not to the extracellular chelation of transferrin-bound 59Fe. The analogues of PIH were more effective in mobilization studies than PIH and DFO, releasing more 59Fe from ferritin as well as from the stroma-mitochondrial membranes in hepatocytes prelabelled using transierrin- 59Fe. Chelator action was dependent on incubation time, concentration, temperature and lipophilicity. Pyridoxal benzoyl hydrazone, the most effective iron chelator, was also the most lipophilic, suggesting that access to cellular iron compartments as well as iron-binding affinity is important in effective iron chelation.

Regulation of Heme Synthesis in Erythroid Cells: Hemin Inhibits Transferrin Iron Utilization but Not Protoporphyrin Synthesis

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2-14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2-14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2-14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Regulation of heme synthesis in erythroid cells: hemin inhibits transferrin iron utilization but not protoporphyrin synthesis

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.