Novel 4-chlorophenoxyacetate dioxygenase-mediated phenoxyalkanoic acid herbicides initial catabolism in Cupriavidus sp. DL-D2.

Microbial metabolism is an important driving force for the elimination of 4-chlorophenoxyacetic acid residues in the environment. The α-Ketoglutarate-dependent dioxygenase (TfdA) or 2,4-D oxygenase (CadAB) catalyzes the cleavage of the aryl ether bond of 4-chlorophenoxyacetic acid to 4-chlorophenol, which is one of the important pathways for the initial metabolism of 4-chlorophenoxyacetic acid by microorganisms. However, strain Cupriavidus sp. DL-D2 could utilize 4-chlorophenoxyacetic acid but not 4-chlorophenol for growth. This scarcely studied degradation pathway may involve novel enzymes that has not yet been characterized. Here, a gene cluster (designated cpd) responsible for the catabolism of 4-chlorophenoxyacetic acid in strain DL-D2 was cloned and identified, and the dioxygenase CpdA/CpdB responsible for the initial degradation of 4-chlorophenoxyacetic acid was successfully expressed, which could catalyze the conversion of 4-chlorphenoxyacetic acid to 4-chlorocatechol. Then, an aromatic cleavage enzyme CpdC further converts 4-chlorocatechol into 3-chloromuconate. The results of substrate degradation experiments showed that CpdA/CpdB could also degrade 3-chlorophenoxyacetic acid and phenoxyacetic acid, and homologous cpd gene clusters were widely discovered in microbial genomes. Our findings revealed a novel degradation mechanism of 4-chlorophenoxyacetic acid at the molecular level.

Machine learning guided rational design of a non-heme iron-based lysine dioxygenase improves its total turnover number.

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based machine-learning algorithm with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40% improvement in the TTN (218±3) as compared to WT LDO (TTN = 160±2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies.

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase.

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts are powerful tools for late-stage C-H functionalization, as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations. Here, we describe the ability of a non-heme iron halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid (Aba) to yield mono-chlorinated, di-chlorinated, and tri-chlorinated products. These chemoselective outcomes are achieved by controlling the loading of 2OG cofactor and SyrB2 biocatalyst. By using a ferredoxin-based biological reductant for electron transfer to the catalytic center of SyrB2, we demonstrate order-of-magnitude enhancement in the yield of tri-chlorinated product that were previously inaccessible using any single halogenase enzyme. We also apply these strategies to broaden SyrB2's reactivity scope to include multi-bromination and demonstrate chemoenzymatic conversion of the ethyl side chain in Aba to an ethylyne functional group. We show how steric hindrance induced by the successive addition of halogen atoms on Aba's C4 carbon dictates the degree of multi-halogenation by hampering C3-C4 bond rotation within SyrB2's catalytic pocket. Overall, our work showcases the synthetic potential of iron halogenases to facilitate multi-C-H functionalization chemistry.

MALDI-mass spectrometry imaging as a new technique for detecting non-heme iron in peripheral tissues via caudal vein injection of deferoxamine.

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC). This method can be performed in anaerobic glove bag settings, requires readily available HPLC instrumentation for subsequent detection, and is adaptable to meet the needs of a variety of substrate affinity measurements.

Non-Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis.

We report the discovery and biosynthesis of new piperazine alkaloids-arizonamides, and their derived compounds-arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non-heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3-alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

The protective association of dairy intake and the adverse impact of iron on gestational diabetes risk.

Background: Gestational diabetes (GDM) is a pregnancy-related glucose intolerance with significant implications for maternal and fetal health. Calcium is essential for insulin secretion and metabolism, while iron intake may also impact GDM. This case-control study was conducted to investigate the relationship between calcium and iron intake with the risk of GDM. Methods: GDM was defined as Fasting Blood Sugar>92mg/dL or 75g Oral-Glucose-Tolerance-Test 120-minutes>153mg/dL. A 168-Item food-frequency-questionnaire was used to collect dietary calcium and iron intake from 24-40 weeks of gestation. The impact of total iron, red, processed/unprocessed meat consumption, calcium, and dairy intake on GDM were investigated. Results: A total of 229 GDM and 205 non-GDM women (18-45 years) participated. GDM group had higher pre-pregnancy weight, weight gain, and pre-pregnancy BMI. Across all models, GDM risk significantly increased in the third and fourth quartiles of iron intake. The fourth quartile had an Odds Ratio (OR) of 2.68 (CI 95%, 4.89-1.56; P<0.001) compared to the reference. Heme-iron consumption in the fourth quartiles increased GDM risk. In the second calcium intake model, ORs for the second, third, and fourth quartiles were 0.51 (CI 95%, 0.91-0.25), 0.43 (CI 95%, 0.77-0.24), and 0.35 (CI 95%, 0.63-0.19), respectively (P<0.001 all), reducing GDM risk by 50-65% compared to the first quartile. Dairy consumption in all quartiles of the first and second models was associated with lower GDM risk. Conclusions: Consumption of heme-iron through red and processed meat associated with an increased chance of developing GDM. Dairy intake reduces the chances of developing GDM in pregnant women.

Oxidized myoglobin: Revealing new perspectives and insights on factors affecting the water retention of myofibrillar proteins.

The impact of oxidized myoglobin (Mb) on myofibrillar protein (MP) oxidation and water retention was investigated. Results showed that the oxidation of Mb increased with increasing concentration of oxidized linoleic acid (OLA). In the presence of 100 mmol/L OLA, hemin iron decreased by 62.07 % compared to the control group. Further investigation showed that mild oxidation of Mb (≤10 mmol/L OLA) increased the water retention and the absolute value of the zeta potential of MP, whereas excessive oxidation (>10 mmol/L OLA) decreased these properties. With the increase of Mb oxidation, the carbonyl content in MP increased, and α-helices changed to random helix. And the tertiary structure changed. Pearson correlation analysis suggested that oxidized Mb affected the water retention of MP, which was closely related to hemin iron and non-hemin iron. In conclusion, OLA induced Mb oxidation, further promoted MP oxidation and affected its water retention.

Rebound or Cage Escape? The Role of the Rebound Barrier for the Reactivity of Non-Heme High-Valent FeIV =O Species.

Owing to their high reactivity and selectivity, variations in the spin ground state and a range of possible pathways, high-valent FeIV =O species are popular models with potential bioinspired applications. An interesting example of a structure-reactivity pattern is the detailed study with five nonheme amine-pyridine pentadentate ligand FeIV =O species, including N4py: [(L1 )FeIV =O]2+ (1), bntpen: [(L2 )FeIV =O]2+ (2), py2 tacn: [(L3 )FeIV =O]2+ (3), and two isomeric bispidine derivatives: [(L4 )FeIV =O]2+ (4) and [(L5 )FeIV =O]2+ (5). In this set, the order of increasing reactivity in the hydroxylation of cyclohexane differs from that with cyclohexadiene as substrate. A comprehensive DFT, ab initio CASSCF/NEVPT2 and DLPNO-CCSD(T) study is presented to untangle the observed patterns. These are well reproduced when both activation barriers for the C-H abstraction and the OH rebound are taken into account. An MO, NBO and deformation energy analysis reveals the importance of π(pyr) → π*xz (FeIII -OH) electron donation for weakening the FeIII -OH bond and thus reducing the rebound barrier. This requires that pyridine rings are oriented perpendicularly to the FeIII -OH bond and this is a subtle but crucial point in ligand design for non-heme iron alkane hydroxylation.

Synthesis, Structure and Reactivity of a Mononuclear N,N,O-Bound Fe(II) α-Keto-Acid Complex.

A bulky, tridentate phenolate ligand (ImPh2 NNOtBu ) was used to synthesise the first example of a mononuclear, facial, N,N,O-bound iron(II) benzoylformate complex, [Fe(ImPh2 NNOtBu )(BF)] (2). The X-ray crystal structure of 2 reveals that the iron centre is pentacoordinate (τ=0.5), with a vacant site located cis to the bidentate BF ligand. The Mössbauer parameters of 2 are consistent with high-spin iron(II), and are very close to those reported for α-ketoglutarate-bound non-heme iron enzyme active sites. According to NMR and UV-vis spectroscopies, the structural integrity of 2 is retained in both coordinating and non-coordinating solvents. Cyclic voltammetry studies show that the iron centre has a very low oxidation potential and is more prone to electrochemical oxidation than the redox-active phenolate ligand. Complex 2 reacts with NO to form a S=3 /2 {FeNO}7 adduct in which NO binds directly to the iron centre, according to EPR, UV-vis, IR spectroscopies and DFT analysis. Upon O2 exposure, 2 undergoes oxidative decarboxylation to form a diiron(III) benzoate complex, [Fe2 (ImPh2 NNOtBu )2 (µ2 -OBz)(µ2 -OH)2 ]+ (3). A small amount of hydroxylated ligand was also observed by ESI-MS, hinting at the formation of a high-valent iron(IV)-oxo intermediate. Initial reactivity studies show that 2 is capable of oxygen atom transfer reactivity with O2 , converting methyl(p-tolyl)sulfide to sulfoxide.

Protein3D: Enabling analysis and extraction of metal-containing sites from the Protein Data Bank with molSimplify.

Metalloenzymes catalyze a wide range of chemical transformations, with the active site residues playing a key role in modulating chemical reactivity and selectivity. Unlike smaller synthetic catalysts, a metalloenzyme active site is embedded in a larger protein, which makes interrogation of electronic properties and geometric features with quantum mechanical calculations challenging. Here we implement the ability to fetch crystallographic structures from the Protein Data Bank and analyze the metal binding sites in the program molSimplify. We show the usefulness of the newly created protein3D class to extract the local environment around non-heme iron enzymes containing a two histidine motif and prepare 372 structures for quantum mechanical calculations. Our implementation of protein3D serves to expand the range of systems molSimplify can be used to analyze and will enable high-throughput study of metal-containing active sites in proteins.

Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopy.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Non-heme Iron Single-Atom Nanozymes as Peroxidase Mimics for Tumor Catalytic Therapy.

Single-atom nanozymes (SAzymes) open new possibilities for the development of artificial enzymes that have catalytic activity comparable to that of natural peroxidase (POD). So far, most efforts have focused on the structural modulation of the Fe-N4 moiety to mimic the metalloprotein heme center. However, non-heme-iron POD with much higher activity, for example, HppE, has not been mimicked successfully due to its structural complexity. Herein, carbon dots (CDs)-supported SAzymes with twisted, nonplanar Fe-O3N2 active sites, highly similar to the non-heme iron center of HppE, was synthesized by exploiting disordered and subnanoscale domains in CDs. The Fe-CDs exhibit an excellent POD activity of 750 units/mg, surpassing the values of conventional SAzymes with planar Fe-N4. We further fabricated an activatable Fe-CDs-based therapeutic agent with near-infrared enhanced POD activity, a photothermal effect, and tumor-targeting ability. Our results represent a big step in the design of high-performance SAzymes and provide guidance for future applications for synergistic tumor therapy.

Metformin ameliorates ferroptosis in cardiac ischemia and reperfusion by reducing NOX4 expression via promoting AMPKα.

CONTEXT: Metformin (Met) has a protective effect against cardiac ischemia and reperfusion (I/R) injury. OBJECTIVE: This study uncovered the Met effect on ferroptosis in cardiac I/R. MATERIALS AND METHODS: Sprague-Dawley rats underwent cardiac I/R treatment (ischaemia 30 min; reperfusion 24 h) (I/R group), and administered intravenously with Met (200 mg/kg) (I/R + Met group). Haematoxylin-eosin staining, Prussian blue staining, immunohistochemistry and transmission electron microscope were conducted on cardiac tissues. H9c2 cells underwent oxygen-glucose deprivation/reoxygenation (OGD/R group) and treated by Met (0.1 mM) (OGD/R + Met group). Adenosine monophosphate-activated protein kinase α (AMPKα) siRNA was transfected into OGD/R-induced H9c2 cells. Cell counting kit-8 (CCK-8) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and JC-1 staining were conducted on H9c2 cells. Ferroptosis-related indicators and gene expression were detected by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: In cardiac I/R rat, Met decreased heart and serum MDA, cardiac and serum non-heme iron, and serum CK-MB and LDH (inhibition rate: 50.0%, 48.8%, 47.6%, 29.5%, 30.6% and 34.7%, respectively), relieved cardiac tissue ferroptosis and mitochondria damage, increased fraction shortening and ejection fraction (157.5% and 146.2% on day 28, respectively), up-regulated AMPKα and down-regulated NOX4 in cardiac tissues. In OGD/R-induced H9c2 cells, Met (0.1 mM) increased cell viability (promotion rate: 170.0%), decreased non-heme iron and MDA (inhibition rate: 30.1% and 47.9%, respectively), relieved ferroptosis, up-regulated AMPKα and down-regulated NOX4. AMPKα silencing abrogated these effects of Met on the OGD/R-induced H9c2 cells. DISCUSSION AND CONCLUSIONS: Met shows effectiveness in relieving ferroptosis in cardiac I/R. In the future, Met may be an effective drug for relieving ferroptosis in cardiac I/R patients clinically.

XDH and XO Research and Drug Discovery-Personal History.

The author will outline the research history of the main issues addressed in this paper. The author has worked on this research himself. XDH, which is responsible for purine degradation, is present in various organisms. However, conversion to XO only occurs in mammals. The molecular mechanism of this conversion was elucidated in this study. The physiological and pathological significance of this conversion is presented. Finally, enzyme inhibitors were successfully developed, two of which are used as therapeutic agents for gout. Their wide application potential is also discussed.

Assessment of Heme and Non-Heme Iron Intake and Its Dietary Sources among Adults in Armenia.

Adequate dietary iron (Fe) intake is crucial for preventing Fe-deficient anemia, a recognized global public health concern which is important in Armenia. This study aimed to analyze the intake of Fe, both heme (from animal tissues) and non-heme (more prevalent, but less efficiently absorbed), as well as the Fe dietary sources, among adults in a representative national sample in Armenia. The study was conducted on 1400 individuals aged 18-80 and above, who were enrolled from all regions of Armenia. The Fe intake was assessed through a 24 h dietary recall survey, while Fe occurrence was determined through atomic absorption spectrophotometry (AAS). The results showed a high proportion of adults with a Fe intake lower than the average requirements set by EFSA (65%, 80% and 85% of males, total females and females at fertile age, respectively). Main Fe sources were bread, fruits and vegetables; heme Fe accounted only for <5% of total Fe intake. Compared to males, females had a lower intake of all forms of Fe (p < 0.05). Significant differences were observed in the intake of different forms of Fe between regions (p < 0.05), while the age-group 36-55 years had higher intakes of total Fe. Our data call for comprehensive nutritional security strategies in order to reduce iron deficiency in Armenia, that represents a public health concern.

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation.

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.

Association between gastric cancer and the intake of different types of iron and meats.

BACKGROUND: Heme and non-heme irons are two forms of iron in the diet. Few studies have evaluated the association between heme iron intake and the risk of gastric cancer (GC). We aimed to investigate the association between heme, non-heme and total iron intake and risk of GC in Iran. METHODS: In a hospital-based case-control study, nutritionists interviewed 178 pathologically confirmed GC patients and 276 controls using a valid Diet History Questionnaire. Multiple logistic regression model was used to estimate Odds Ratios (OR) and 95% Confidence Intervals (CIs) for iron intake and risk of GC. RESULTS: Subjects in the highest tertile of total iron intake were 46% less likely to get GC than those in the lowest (OR = 0.54, 95% CI: 0.32-0.92), however, the associations were not significant for intake of heme and non-heme iron. The risk of GC in the highest tertile of total meat intake was 2.51 times higher than the lowest. We found significant associations between GC and chicken (OR = 2.95; 95% CI: 1.66-5.22) and fish intake (OR = 1.89; 95% CI: 1.09-3.27), However, we found no associations between the risk of GC and intake of red meat, salted fish, and liver. CONCLUSION: Total iron intake was associated with a lower risk of GC which could be partly due to the high prevalence of anemia in Iran. Although, we could not find any significant association between the risk of GC and the intake of heme and non-hem iron among the Iranian population.

Estimation of the availability of iron in the school meals of Municipal Centers for Early Childhood Education of a capital city in northeastern Brazil.

The final stage of Fe deficiency is Fe deficiency anaemia, with repercussions for human health, especially in children under 5 years of age. Studies conducted in Brazilian public daycare centres show high prevalence of anaemia. The present study aims to evaluate the availability of Fe in the meals of the Municipal Centers of Early Childhood Education in Maceió. The experimental design comprises selection of algorithms, menu evaluation, calculation of the estimates, comparison between the estimates obtained and the recommendations, and analysis of correlation between meal constituents, and of the concordance between the absorbable Fe estimates. Four algorithms were selected and a monthly menu consisting of 22 d. The correlation analysis showed a moderate positive correlation to animal tissue (AT) v. non-heme iron (r = 0·42; P = 0·04), and negative to AT v. Ca (r = -0·54; P = 0·09) and Ca v. phytates (r = -0·46, P = 0·03). Estimates of absorbable Fe ranged from 0·23 to 0·44 mg/d. The amount of Fe available, unlike the total amount of Fe offered, does not meet the nutritional recommendations on most school days. The Bland-Altman analysis indicated that the Monsen and Balinfty and Rickard et al. showed greater agreement. The results confirm the need to adopt strategies to increase the availability of Fe in school meals.