Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase

Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.     

Formation of free radical metabolites in the reaction between soybean lipoxygenase and its inhibitors. An ESR study

Recent studies showed that soybean lipoxygenase inhibitors like phenidone and nordihydroguaiaretic acid (NDGA) reduce the catalytically active ferric lipoxygenase to its inactive ferrous form. Addition of 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) regenerated the active ferric form. In this paper, it is shown that in such a system the inhibitors are oxidized to free-radical metabolites. Incubation of soybean lipoxygenase and linoleic acid with p-aminophenol, catechol, hydroquinone, NDGA, or phenidone resulted in the formation of the one-electron oxidation products of these compounds. Free-radical formation depended upon the presence of the lipoxygenase and 13-HPOD. The free radicals were detected by ESR spectroscopy, and their structure was confirmed by analysis of the spectra, using a computer correlation technique. These data support the proposed mechanism for the inhibition of lipoxygenase by phenolic antioxidants.     

Interaction of alpha-tocopherol with iron: antioxidant and prooxidant effects of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron

The dual functions of alpha-tocopherol in the oxidation of lipids in aqueous dispersions in the presence of iron were studied, aiming specifically at elucidating the effect of interaction between alpha-tocopherol and iron. Ferrous ion decomposed hydroperoxide rapidly and induced the free radical chain oxidation of soybean phosphatidylcholine liposomes. alpha-Tocopherol acted primarily as a radical scavenger in the oxidation induced by ferrous ion and acted as an antioxidant. Ferric ion decomposed hydroperoxide much more slowly than ferrous ion, but it also induced the oxidation of liposomal membranes. alpha-Tocopherol incorporated into artificial liposomal membranes reduced ferric ion rapidly to give more reactive ferrous ion, and alpha-tocopherol acted either as an antioxidant or as a prooxidant depending on the experimental conditions. When alpha-tocopherol was depleted by the interaction with ferric ion, it acted solely as a prooxidant, whereas if some alpha-tocopherol remained, it acted as an antioxidant. On the other hand, alpha-tocopherol residing in the intact erythrocyte membranes did not reduce ferric ion in the aqueous region.     

The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.     

Generation of Oxygen Radicals from Iron Complex of Orellanine, A Mushroom Nephrotoxin; Preliminary ESR and Spin-Trapping Stukes

Orellanine, [2,2'-bipyridine]-3,3',4,4'-tetrol-l,I'-dioxide, is the toxin responsible for the lethal nephrotoxicity of some Cortinarius mushrooms. Our present ESR and spin-trapping studies of the redox properties of the system of non-illuminated orellanine, ferrous iron and dioxygen contribute to understanding the molecular mechanism of its toxicity. UV-visible spectrophoto-metry, cyclic voltammetry and ESR in frozen medium showed the formation of a wine-red tris complex, Fe(III)Or₃. This ferric complex is easily reducible (E_P =-565 mV vs Ag/AgCl/3M KCl at pH7), involving a one-electron reversible process. Spin-trapping using DMPO is employed to detect the generation of super-oxide anion and hydroxyl radicals. The instantaneous one-electron oxidation of ferrous ions in the presence of the toxin under air is concomitant with dioxygen consumption as supported by dioxygen consumption. GSH involves the toxin and ferrous ions under air in a redox cycling process resulting in the production of glutathionyl and oxygen free radicals, observed for the first time with an iron complex of a mushroom toxin. In most cases, EDTA is not able to prevent the Fe(III)Or₃ and radical formation. The ortho-dihydroxylated groups borne by the di-N-oxidized bipyridine structure and not the bipyridine structure itself, are responsible for the formation of a stable ferric complex at pH 7, as they are for the generation of an apparently stable ortho-semiquinone anion radical. These one-electron mechanisms may play a major role in some of the known toxic effects of orellanine.     

Hydroxyl radical formation from the auto-reduction of a ferric citrate complex.

When a ferric citrate complex is prepared from citric acid and ferric chloride, and the pH value left unchanged, a reduction of the iron moiety takes place. Within several hours a substantial yield of ferrous ions can be detected in the solution. When placed in a phosphate buffer pH 7.0 with a suitable detector molecule, oxidative damage to the detector molecule can be observed. Thus, deoxyribose is degraded with the release of thiobarbituric acid-reactive material and benzoate is hydroxylated to form fluorescent dihydroxy products. Damage can be prevented by scavengers of the hydroxyl radical such as mannitol, formate the thiourea, by catalase and by the protein caeruloplasmin, suggesting that Fenton chemistry occurs leading to the formation of hydroxyl radicals.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

Role of K(+) binding residues in stabilization of heme spin state of Leishmania major peroxidase

The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K(+) site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV-visible spectrophotometry, we show that the removal of K(+) binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K(+) site double mutants in the presence of H(2)O(2). Similarly electron donor oxidation rate was two orders less for the K(+) site double mutants compared to the wild type. These data show that K(+) functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.     

Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa

Neopterin and 7,8-dihydroneopterin, two compounds which are secreted by activated macrophages, have been shown to interfere with radicals generated by cellular and certain chemical systems. Reduced pterins were reported to scavenge whereas aromatic pterins promoted or reduced radical mediated reactions or had no effect. However, recently it was found that high concentrations of 7, 8-dihydroneopterin enhanced luminol dependent chemiluminescence and T-cell apoptosis, suggesting an enhancement of free radical formation. In this study hydroxylation of salicylic acid was used for detection of hydroxyl radicals. It is shown that in solutions of 7,8-dihydroneopterin hydroxyl radicals were formed in the absence of any radical source. The presence of EDTA chelated iron enhanced hydroxyl radical formation. Whereas the addition of iron accelerated the hydroxylation reaction, 7,8-dihydroneopterin was responsible for the amount of hydroxylation products. In the presence of superoxide dismutase or catalase, as well as by helium purging, hydroxylation was inhibited. Our data suggest that in solutions of 7, 8-dihydroneopterin superoxide radicals are generated which are converted to hydroxyl radicals by Fenton or Haber-Weiss type reactions. While superoxide might be generated during autoxidation of ferrous iron, dihydroneopterin seems to be involved in regeneration of ferrous iron from the ferric form.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Effects of 2,3-diketo-L-gulonic acid on the oxidation of yolk lipoprotein

2,3-Diketo-L-gulonic acid (DKG) is an important intermediate product of oxidative degradation of L-ascorbic acid (AsA) in both biological and food systems, but the physiological function of DKG is still unclear. In this study, it was found that DKG had a strong antioxidative effect on copper-dependent oxidative modification of yolk lipoprotein (YLP), on the basis of both the decreased electrophoretic mobility and longer lag time of conjugated diene formation in a concentration-dependent manner. DKG is known to be very unstable and easily converts into two delta-lactones of DKG, the 3,4-enediol form of DKG delta-lactone (3,4-DKGL) and 2,3-enediol form of DKG delta-lactone (2,3-DKGL) depending on both pH and temperature. 3,4-DKGL was thought to be the first degradation product of DKG and could play an antioxidative role in the oxidation of lipoproteins induced by copper ion or peroxyl radicals in neutral aqueous solution.     

The effect of ferric ion on the rate of ferrous oxidation by Thiobacillus ferrooxidans

 In this study, the effect of ferric ion and cell concentrations on the oxidation of ferrous ion by T. ferrooxidans was investigated. Ferric ions competitively inhibited ferrous ion oxidation by the bacteria. The inhibitory effect of ferric ion was, however, reduced by increasing cell concentration. The apparent ferric ion inhibition constant did not change with increasing cell concentration. The ferrous ion oxidation kinetics in the absence and presence of ferric ion changes from the standard Michaelis-Menten type at low cell concentrations to pseudo-first-order kinetics at high cell concentration. Received: 8 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

Reactions of the NAD radical with higher oxidation states of horseradish peroxidase

The reactions of the NAD radical (NAD.) with ferric horseradish peroxidase and with compounds I and II were investigated by pulse radiolysis. NAD. reacted with the ferric enzyme and with compound I to form the ferrous enzyme and compound II with second-order rate constants of 8 X 10(8) and 1.5 X 10(8) M-1 s-1, respectively, at pH 7.0. In contrast, no reaction of NAD. with native compound II at pH 10.0 nor with diacetyldeutero-compound II at pH 5.0-8.0 could be detected. Other reducing species generated by pulse radiolysis, such as hydrated electron (eaq-), superoxide anion (O2-), and benzoate anion radical, could not reduce compound II of the enzyme to the ferric state, although the methylviologen radical reduced it. The results are discussed in relation to the mechanism of catalysis of the one-electron oxidation of substrates by peroxidase.     

Nonenzymatic reduction of ferric leghemoglobin

Ferric leghemoglobin isolated from soybean root nodules was reduced nonenzymatically to ferrous leghemoglobin in vitro at pH 5.2 using either 1.0 mM NADH or NADPH as the reductant. In the pH range of 5.2 to 7.0, the highest rates of reduction occurred below pH 6.5 with a maximum rate observed at pH 5.2. Rates of nonenzymatic ferric leghemoglobin reduction above pH 6.5 or at reduced-pyridine nucleotide concentrations below 0.4 mM were insignificant. Oxygen was required for the nonenzymatic reduction. Inhibition of ferric leghemoglobin reduction by superoxide dismutase and catalase indicated that superoxide and hydrogen peroxide may be intermediates in the reaction.     

Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.     

Production of hydroxyl radical by the synergistic action of fungal laccase and aryl alcohol oxidase

A mechanism for the production of hydroxyl radical (*OH) during the oxidation of hydroquinones by laccase, the ligninolytic enzyme most widely distributed among white-rot fungi, has been demonstrated. Production of Fenton reagent (H2O2 and ferrous ion), leading to *OH formation, was found in reaction mixtures containing Pleurotus eryngii laccase, lignin-derived hydroquinones, and chelated ferric ion. The semiquinones produced by laccase reduced both ferric to ferrous ion and oxygen to superoxide anion radical (O2*-). Dismutation of the latter provided the H2O2 for *OH generation. Although O2*- could also contribute to ferric ion reduction, semiquinone radicals were the main agents accomplishing the reaction. Due to the low extent of semiquinone autoxidation, H2O2 was the limiting reagent in Fenton reaction. The addition of aryl alcohol oxidase and 4-methoxybenzyl alcohol (the natural H2O2-producing system of P. eryngii) to the laccase reaction greatly increased *OH generation, demonstrating the synergistic action of both enzymes in the process.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans

A chemo-biochemical process using Thiobacillus ferrooxidans for desulphurization of gaseous fuels and emissions containing hydrogen sulphide (H2S) has been developed. In the first stage, H2S present in fuel gas and emissions is selectively oxidized to elemental sulphur using ferric sulphate. The ferrous sulphate produced in the first stage of the process is oxidized to ferric sulphate using Thiobacillus ferrooxidans for recycle and reuse in the process. The effects of process variables, temperature, pH, total dissolved solids (TDS), elemental sulphur, ferric and magnesium ions on bio-oxidation of ferrous ions to ferric ions were investigated using flask culture experiments. The bio-oxidation of ferrous ions to ferric ions could be achieved efficiently in the temperature range of 20(+/-1)-44(+/-1) degrees C. A pH range of 1.8(+/-0.02)-2.2(+/-0.02) was optimum for the growth of culture and effective bio-oxidation of ferrous ions to ferric ions. The effect of TDS on bio-oxidation of ferrous ions indicated that a preacclimatized culture in a growth medium containing high dissolved solid was required to achieve effective bio-oxidation of ferrous ions. Elemental sulphur ranging from 1000 to 100,000 mg/l did not have any effect on efficiency of ferrous ion oxidation. The efficiency of bio-oxidation of ferrous ions to ferric ions was not affected in the presence of ferric ions up to a concentration of 500 mg/l while 3 mg/l of magnesium ion was optimal for achieving effective bio-oxidation.     

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum.

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidine's effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.     

Humic acid and iron uptake by plants

Summary The behaviour of ferric EDTA and ferric citrate in nutrient solution and their interaction with humic acid was investigated at various hydrogen ion concentrations using the technique of membrane ultrafiltration to separate small iron species from high molecular weight products of hydrolysis and to estimate the binding of iron by humic acid. Ferric EDTA was found to be of small molecular size at all pH values between 5.0 and 7.0 whilst ferric citrate solutions contained an increasing proportion of high molecular weight material as pH was increased from 5.0 to 7.0. Some iron present in solutions of both ferric EDTA and ferric citrate was bound by humic acid at all pH values from 5.0 to 7.0. Studies were also made of the uptake of iron by wheat roots from nutrient solutions containing either ferric EDTA or ferric citrate and of the effect of humic acid on uptake. More iron was absorbed from ferric EDTA than from ferric citrate at all pH values. Increasing pH between 5.0 and 7.0 resulted in a progressive decrease in the uptake of iron in both cases. The presence of humic acid depressed iron absorption from both solutions at all pH values.