Acute Copper Supplementation Does Not Inhibit Non-Heme Iron Bioavailability in Humans

To measure the effect of acute copper (Cu) administration, given as an aqueous solution, on the absorption of iron (Fe), 29 healthy adult women participated in two iron absorption studies. Subjects received 0.5 mg of Fe, as ferrous sulfate, alone or with Cu, as copper sulfate, at 0.5:1, 1:1, or 2:1 Cu/Fe molar ratios (study I) or at 4:1, 6:1, or 8:1 Cu/Fe molar ratios (study II) as an aqueous solution on days 1, 2, 14, and 15 of the study. Fe absorption was assessed by erythrocyte incorporation of iron radioisotopes ⁵⁵Fe and ⁵⁹Fe. Geometric mean (range ± SD) absorption of Fe alone or at 0.5:1, 1:1, 2:1 Cu/Fe molar ratios were 34.4% (17.3–68.5%), 40.9% (24.9–67.2%), 48.3% (24.8–94.1%), and 50.2% (25.3–99.5%), respectively (ANOVA, p = 0.12). Geometric mean (range ± SD) absorption of Fe alone or at 4:1, 6:1, 8:1 Cu/Fe molar ratios were 28.7% (12.1–67.9%), 21.5% (6.5–71.5%), 29.6% (10.3–85.4%), and 36.5% (18.3–73.1%), respectively (ANOVA, p = 0.16). In conclusion, combined Cu and Fe administration in an aqueous solution does not inhibit Fe bioavailability. This information could help in the design of rational guidelines for copper and iron supplementation programs. Our results support the hypothesis that divalent metal transporter 1 is not physiologically relevant for copper absorption in humans.     

Acute inhibition of iron bioavailability by zinc: studies in humans

Iron (Fe) and zinc (Zn) deficiencies constitute two of the most important nutritional and public health problems affecting developing countries. Combined supplementation or fortification with Zn and Fe are strategies that can be used to improve the Zn and Fe status of a population. However, there is concern about potential negative interactions between these two micronutrients due to a competitive binding to DMT1 and Zip14 transporter. Studies performed in humans have shown an inhibitory effect of Zn on Fe absorption when both minerals are given together as a solution in fasting conditions. We found that at low doses of iron (0.5 mg) the threshold for the inhibition of iron bioavailability was at a Zn:Fe wt/wt ratio ≥5.9:1, whereas at higher doses of Fe (10 mg) this inhibition occurred at 1:1 Zn:Fe wt/wt ratio. This differential response could be explained by the variation in the abundance of both cations as they compete for a limited number of shared transporters at the enterocyte. Conflicting results have been obtained when this interaction was studied in different food matrices. A negative interaction was not observed when Fe and Zn were provided in a composite hamburger meal, premature formula, human milk, or cow milk. A decrease on Fe absorption was observed in only 1 of 3 studies when Fe and Zn were supplied in wheat flour. The possibility of a negative interaction should be considered for supplementation or fortification programs with both microminerals.     

Bioavailability of Stabilised Ferrous Gluconate with Glycine in Fresh Cheese Matrix: a Novel Iron Compound for Food Fortification

Iron fortification of foods continues to be one of the preferred ways of improving the iron status of the population. Dairy product is a common product in the diet; therefore, it is a plausible vehicle for iron fortification. This study aims to investigate the bioavailability of ferrous gluconate stabilised with glycine (FGSG) in a fresh cheese fortified with zinc. The iron bioavailability of fresh cheese fortified with either FGSG and with or without zinc and FGSG in aqueous solution and a water solution of ferrous ascorbate (reference dose) was studied using double radio iron (⁵⁵Fe and ⁵⁹Fe) erythrocyte incorporation in 15 male subjects. All subjects presented with normal values for iron status parameters. The geometric mean of iron bioavailability for the water solution of FGSG was 38.2 %, adjusted to 40 % from reference doses (N.S.). Iron bioavailability in fresh cheese fortified with Ca and Zn was 15.4 % and was 23.1 % without Zn, adjusted to 40 % from reference doses (N.S.). The results of the present study show that the novel iron compound ferrous gluconate stabilised with glycine in a fresh cheese matrix is a good source of iron and can be used in iron fortification programmes.     

Dephytinisation with Intrinsic Wheat Phytase and Iron Fortification Significantly Increase Iron Absorption from Fonio (Digitaria exilis) Meals in West African Women

Low iron and high phytic acid content make fonio based meals a poor source of bioavailable iron. Phytic acid degradation in fonio porridge using whole grain cereals as phytase source and effect on iron bioavailability when added to iron fortified fonio meals were investigated. Grains, nuts and seeds collected in Mali markets were screened for phytic acid and phytase activity. We performed an iron absorption study in Beninese women (n = 16), using non-dephytinised fonio porridge (FFP) and dephytinised fonio porridge (FWFP; 75% fonio-25% wheat), each fortified with ⁵⁷Fe or ⁵⁸Fe labeled FeSO₄. Iron absorption was quantified by measuring the erythrocyte incorporation of stable iron isotopes. Phytic acid varied from 0.39 (bambara nut) to 4.26 g/100 g DM (pumpkin seed), with oilseeds values higher than grains and nuts. Phytase activity ranged from 0.17±1.61 (fonio) to 2.9±1.3 phytase unit (PU) per g (whole wheat). Phytic acid was almost completely degraded in FWFP after 60 min of incubation (pH≈5.0, 50°C). Phytate∶iron molar ratios decreased from 23.7∶1 in FFP to 2.7∶1 in FWFP. Iron fortification further reduced phytate∶iron molar ratio to 1.9∶1 in FFP and 0.3∶1 in FWFP, respectively. Geometric mean (95% CI) iron absorption significantly increased from 2.6% (0.8–7.8) in FFP to 8.3% (3.8–17.9) in FWFP (P<0.0001). Dephytinisation of fonio porridge with intrinsic wheat phytase increased fractional iron absorption 3.2 times, suggesting it could be a possible strategy to decrease PA in cereal-based porridges.     

Inhibition of zinc absorption by iron depends on their ratio

Previous studies upon zinc-iron interactions gave conflicting results that could come from differences in protocol design or in trace element status of subjects. The present work assessed the influence of zinc : iron ratio and iron deficiency upon zinc absorption. The digestive absorption of zinc sulphate (100 mol Zn/l) in presence of iron gluconate was studied in perfused jejunal loops (n = 6/group) of normal rats (range 0–1000 mol Fe/l) and iron deficient rats (200–750 mol Fe/l). In normal rats no significant iron inhibition on zinc absorption occurred at Fe:Zn ratio below 2:1. At higher ratios zinc uptake and net absorption decreased significantly (p<0.05). Between 2:1 and 5:1 a dose dependent inhibition of zinc absorption occurred and reached a plateau beyond this ratio. In iron deficient animals no changes in zinc uptake, mucosal retention and absorption compared to normal animals occurred at ratio 2:1. At higher ratios differences were observed at every zinc absorption step except for mucosal retention at 7.5:1 ratio.

Iron-zinc interactions depend on their ratio and on previous trace elements status of subjects. Due to the wide and unknown variations that were likely to occur between the subjects of previous human and experimental studies, these results could explain some of the discrepancies between their results.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Long-term effects of iron: Zinc interactions on growth in rats

The influence of iron (Fe) on the bioavailability and functional status of zinc (Zn) was studied in young rats using metabolic balances and tissue dosages, which were compared to growth. Diets supplied adequate intakes of Fe (45 and 300 mg/kg diet) and Zn (14 and 45 mg/kg) for 2 mo. Two metabolic balance determinations were performed that were correlated for Zn and Fe during the first and the last weeks of the study. A significant effect of Fe supply, but not of Zn was displayed on Fe absorption; both Fe and Zn diet concentrations had a significant influence on Zn absorption. Fe and Zn organ contents were significantly correlated with the amount absorbed during the two metabolic balances. There was a positive correlation between liver and muscle Fe and Fe absorption, and Fe absorption and muscle Zn, as well as a negative one with liver Zn; a positive correlation was displayed between Zn absorption and Zn organ content. No correlation was found between Zn absorption and Fe tissue content. Growth was correlated with Zn, but not with Fe absorption during both balances. A positive correlation was displayed between growth and Zn liver content, and a negative one with Fe liver content. Care must be taken to give growing subjects balanced diets or supplementation, since the negative interactions between these trace elements are likely to persist as long as the diet is given.     

Preventive Effects of Zinc Against Psychological Stress-Induced Iron Dyshomeostasis, Erythropoiesis Inhibition, and Oxidative Stress Status in Rats

Psychological stress (PS) could cause decreased iron absorption and iron redistribution in body resulting in low iron concentration in the bone marrow and inhibition of erythropoiesis. In the present study, we investigated the effect of zinc supplementation on the iron metabolism, erythropoiesis, and oxidative stress status in PS-induced rats. Thirty-two rats were divided into two groups randomly: control group and zinc supplementation group. Each group was subdivided into two subgroups: control group and PS group. Rats received zinc supplementation before PS exposure established by a communication box. We investigated the serum corticosterone (CORT) level; iron apparent absorption; iron contents in liver, spleen, cortex, hippocampus, striatum, and serum; hematological parameters; malondialdehyde (MDA); reduced glutathione (GSH); and superoxide dismutase (SOD). Compared to PS-treated rats with normal diet, the PS-treated rats with zinc supplementation showed increased iron apparent absorption, serum iron, hemoglobin, red blood cell, GSH, and SOD activities; while the serum CORT; iron contents in liver, spleen, and regional brain; and MDA decreased. These results indicated that dietary zinc supplementation had preventive effects against PS-induced iron dyshomeostasis, erythropoiesis inhibition, and oxidative stress status in rats.     

Inhibition of iron and copper uptake by iron, copper and zinc

Interactions of micronutrients can affect absorption and bioavailability of other nutrients by a number of mechanisms. In aqueous solutions, and at higher uptake levels, competition between elements with similar chemical characteristics and uptake process can take place. The consequences of these interactions may depend on the relative concentrations of the nutrients. In this work, we measure the effects of increasing concentrations of iron, zinc, and copper on iron and copper uptake in Caco-2 cells. Intracellular Fe or Cu levels were affected by incubating with increased concentrations of metals. However, when the cells already had different intracellular metal concentration, the uptake of Fe or Cu was nor affected. In competition studies, we showed that Cu and Zn inhibited Fe uptake, and while Fe inhibited Cu uptake, Zn did not. When the three metals were given together (1:1:1 ratio), Fe or Cu uptake was inhibited approximately 40%. These results point to a potential risk in the absorption and bioavailability of these minerals by the presence of other minerals in the diet. This aspect must be considered in food supplementation and fortification programs.     

Iron bioavailability of common beans (Phaseolus vulgaris L.) intrinsically labeled with 59Fe

A radiobioassay was performed in rats with or without iron depletion to evaluate the iron bioavailability of diets enriched with common beans and with “multimixture”, a nutritional supplement based on parts of foods that are not usually eaten. The full-body ⁵⁹Fe level was determined after 5 h, the absorbed ⁵⁹Fe level was determined after 48 h, and the amount of ⁵⁹Fe retained was determined after 7 days. Iron bioavailability was assessed by the full-body radioactivity of the animals, determined using a solid scintillation detector. The iron bioavailability of common beans was higher in the iron-depleted animals (55.7%) than in the non-depleted animals (25.12%) because of the higher absorption rate in the iron-depleted animals. The multimixture did not influence dietary iron bioavailability. In addition, the iron bioavailability of common beans was similar to that observed in the standard source of iron for Wistar rats. Hence, common beans may be considered an adequate dietary iron source because of its high bioavailability.     

Effect of Increasing Levels of Zinc Fortificant on the Iron Absorption of Bread Co-Fortified with Iron and Zinc Consumed with a Black Tea

Iron (Fe) and zinc’s (Zn) interaction at the absorptive level can have an effect on the success of co-fortification of wheat flour with both minerals on iron deficiency prevention. The aim of the study was to determine the effect of increasing levels of zinc fortificant on the iron absorption of bread co-fortified with iron and zinc consumed with a black tea. Twelve women aged 33–42 years participated in the study. They received on four different days 200 mL of black tea and 100 g of bread made with wheat flour (70 % extraction) fortified with either 30 mg Fe/kg alone, as ferrous sulfate (A), or with the same Fe-fortified flour, but with graded levels of Zn, as zinc sulfate: 30 mg/kg (B), 60 mg/kg (C), or 90 mg/kg (D). Fe radioisotopes (⁵⁹Fe and ⁵⁵Fe) of high specific activity were used as tracers, and Fe absorption iron was measured by the incorporation of radioactive Fe into erythrocytes. The geometric mean and range of ±1 SD of Fe absorption were as follows: A?=?6.5 % (2.2–19.3 %), B?=?4.6 % (1.0–21.0 %), C?=?2.1 % (0.9–4.9 %), and D?=?2.2 % (0.7–6.6 %), respectively; ANOVA for repeated measures F?=?10.9, p?<?0.001 (Scheffè’s post hoc test: A vs. C, A vs. D, B vs. C, and B vs. D; p?<?0.05). We can conclude that Fe absorption of bread made from low-extraction flour fortified with 30 mg/kg of Fe, as ferrous sulfate, and co-fortified with zinc, as zinc sulfate consumed with black tea is significantly decreased at a zinc fortification level of ≥60 mg/kg flour.     

Long-term zinc and iron supplementation in children of short stature: effect of growth and on trace element content in tissues.

We evaluated the effect of one year of supplementation with iron plus zinc (12 mg/day of Fe+++ and 12.5 mg/day of Zn++), zinc alone (12.5 mg/day of Zn++) and placebo on growth and on the iron, zinc, copper and selenium tissue contents in 30 well-selected children of short stature (16 M and 14 F; 4-11 years old). Before and after supplementation, we measured the concentrations of iron, transferrin, ferritin, zinc and copper in serum, of zinc in erythrocytes and leukocytes, and of zinc, copper and selenium in hair, as well as glutathione peroxidase activity in erythrocytes. Before supplementation, ferritin and serum, erythrocyte and hair zinc contents were significantly lower than in age-matched controls, while the other measured indices were in the normal range. Iron plus zinc supplementation caused an improvement in growth rate in all subjects, i.e., the median Z-score increased from -2.22 +/- 0.45 to -0.64 +/- 0.55; (p < 0.01). In the zinc-supplemented group, only the subjects whose ferritin levels were higher than 20 ng/L before supplementation showed a similar improvement of growth rate. Iron plus zinc supplementation could be a reasonable treatment in short, prepubertal children affected by marginal zinc and iron deficiency.     

Pectin Esterification Degree in the Bioavailability of Non-heme Iron in Women

Pectins are a type of soluble fiber present in natural and processed foods. Evidence regarding the effect of esterification degree of pectins on iron absorption in humans is scarce. In the present study, the effect of pectins with different degrees of esterification on non-heme iron absorption in women was evaluated. A controlled experimental study was conducted with block design, involving 13 apparently healthy, adult women. Each subject received 5 mg Fe (FeSO₄) without pectin (control) or accompanied by 5 g citrus pectin, two with a low degree of esterification (27 and 36%), and one with a high degree of esterification (67 to 73%), each on different days. Each day, the 5 mg Fe doses were marked with radioactive ⁵⁹Fe or ⁵⁵Fe. Radioactivity incorporated into erythrocytes was determined in blood samples 14 days after the marked Fe doses were consumed. On days 18 and 36 of study, 30 and 20 mL blood samples were obtained, respectively, and blood sample radioactivity incorporated into erythrocytes was determined. Body iron status was determined from blood taken on day 18. Whole body blood volume was estimated for calculate iron bioavailability; it was assumed that 80% of absorbed radioactivity was incorporated into the Hb. All women participants signed an informed consent of participation at baseline. Iron bioavailability (mean geometric ±1 SD) alone (control) was 18.2% (12.3–27.1%), iron + pectin27 was 17.2% (10.2–29.2%), iron + pectin36 was 15.3% (9.5–24.6%), and iron + pectin67 was 19.5% (10.0–38.0%). No statistically significant differences between iron bioavailability (repeated measures ANOVA, p = 0.22) were observed. Pectin esterification degree does not influence the bioavailability of non-heme iron in women.     

Effect of Helicobacter pylori Infection on Iron Absorption in Asymptomatic Adults Consuming Wheat Flour Fortified with Iron and Zinc

Helicobacter pylori infection could impair iron absorption from fortified products. The objective of the study was to determine the effect of H. pylori infection on iron absorption from asymptomatic adults consuming wheat flour fortified with iron and zinc. The (13)C urea breath test was used to assess H. pylori infection. Twenty-four H. pylori-positive and 26 H. pylori-negative volunteers completed the study. On day?1, the subjects were randomized to receive for breakfast bread fortified with either ferrous sulfate and zinc sulfate or ferrous fumarate and zinc oxide. Bread fortified with ferrous sulfate was labeled with (59)Fe as sulfate, and bread fortified with ferrous fumarate was labeled with (55)Fe as fumarate. On day?3, they received the other type of bread, with the respective tracers. On days?18-23, a proton pump inhibitor was administered to all subjects. On day?24, all subjects received bread fortified with ferrous fumarate and zinc oxide labeled with (55)Fe as fumarate. H. pylori prevalence was 77.6%. The geometric mean (±1?SD) of iron absorption was significantly higher for ferrous sulfate than fumarate (6.9?±?2.9% vs. 0.5?±?3.5%, p?相似文献   

Iron incorporation and post-malaria anaemia

Background

Iron supplementation is employed to treat post-malarial anaemia in environments where iron deficiency is common. Malaria induces an intense inflammatory reaction that stalls reticulo-endothelial macrophagal iron recycling from haemolysed red blood cells and inhibits oral iron absorption, but the magnitude and duration of these effects are unclear.

Methodology/Principal Findings

We examined the red blood cell incorporation of oral administered stable isotopes of iron and compared incorporation between age matched 18 to 36 months old children with either anaemia post-malaria (n = 37) or presumed iron deficiency anaemia alone (n = 36). All children were supplemented for 30 days with 2 mg/kg elemental iron as liquid iron sulphate and administered ⁵⁷Fe and ⁵⁸Fe on days 1 and 15 of supplementation respectively. ⁵⁷Fe and⁵⁸Fe incorporation were significantly reduced (8% vs. 28%: p<0.001 and 14% vs. 26%: p = 0.045) in the malaria vs. non-malaria groups. There was a significantly greater haemoglobin response in the malaria group at both day 15 (p = 0.001) and 30 (p<0.000) with a regression analysis estimated greater change in haemoglobin of 7.2 g/l (s.e. 2.0) and 10.1 g/l (s.e. 2.5) respectively.

Conclusion/Significance

Post-malaria anaemia is associated with a better haemoglobin recovery despite a significant depressant effect on oral iron incorporation which may indicate that early erythropoetic iron need is met by iron recycling rather than oral iron. Supplemental iron administration is of questionable utility within 2 weeks of clinical malaria in children with mild or moderate anaemia.     

Effect of Increasing Concentrations of Zinc on the Absorption of Iron from Iron-Fortified Milk

The cofortification of milk with iron (Fe) and zinc (Zn) is a strategy used to prevent these deficiencies during childhood. Given that Zn can negatively interact with iron in aqueous solutions, the objective of the present study was to determine the effect of Zn on Fe absorption of milk fortified with Fe and Zn. Twenty-eight women between 33 and 47?years of age, with contraception and a negative pregnancy test, participated in one of two absorption studies. They received on four different days, after an overnight fast, 200?mL of milk (26?% fat) fortified with 10?mg Fe/L, as (A) ferrous sulfate, or the same milk but with graded doses of added Zn, as Zn sulfate of (B) 5, (C) 10, and (D) 20?mg/L (study 1, n?=?15). In study 2 (n?=?13), subjects received the same milk formulations, but these were also fortified with ascorbic acid (70?mg/L). Milk was labeled with radioisotopes ⁵⁹Fe or ⁵⁵Fe, and the absorption of iron was measured by erythrocyte incorporation of radioactive Fe. The geometric mean and range of ±1 SD of Fe absorption in study 1 were as follows: formula A?=?6.0?% (2.8?C13.0?%); B?=?6.7?% (3.3?C13.6?%); C?=?5.4?% (2.2?C13.2?%); and D?=?5.2?% (2.8?C10.0?%) (ANOVA for repeated measures, not significant). For study 2, data are as follows: 8.2?% (3.6?C18.7?%); B?=?6.4?% (2.5?C16.4?%); C?=?7.7?% (3.2?C18.9?%); and D?=?5.2 (1.8?C14.8?%) (ANOVA for repeated measures, not significant). In conclusion, according to the results from this study, it appears that the addition of zinc up to 20?mg/L does not significantly inhibit iron absorption from milk fortified with 10?mg/L of iron.     

Iron Specifically Protects Corn Protoplasts from T-Toxin of Cochliobolus heterostrophus

Ferric ion reduced the damaging effects of T-toxin, a series of linear β-polyketols produced by the pathogenic fungus Cochliobolus heterostrophus, on leaf mesophyll protoplasts from susceptible T-cytoplasm corn. Of nine metals tested, only ferric and ferrous ions had this effect. Despite the presence of 12 available oxygen atoms in each T-toxin molecule, there was no evidence for the formation of an aqueous Fe²⁺- or Fe³⁺-T-toxin complex. The protective effect of iron was eliminated by a molar excess of EDTA. Iron had no effect on the sensitivity of T-cytoplasm mitochondria to T-toxin, even at a 1000-fold molar excess, nor did it protect roots of T-cytoplasm corn seedlings from inhibition by T-toxin. The mechanism by which iron specifically protects protoplasts from T-toxin is not understood, but time lapse experiments suggest that iron acts on some intracellular site to modify T-toxin sensitivity and not on a transport system at the cell surface.     

Potential role of in vitro iron bioavailability studies in combatting iron deficiency: a study of the effects of phosvitin on iron mobilization from pinto beans

Iron deficiency and iron overload affect one billion people worldwide. Treatment of iron malnutrition can be enhanced by an understanding of iron bioavailability from the diet. We have focused on the development of in vitro methods for determining iron bioavailability in the hopes of providing both an understanding of the chemical basis leading to the inhibition or enhancement of iron absorption and the provision of methodologies which will allow nutritionists around the world to ascertain iron bioavailability of local foods and food combinations. The study reported here focuses on the effects of phosvitin, a suspected inhibitor of iron absorption found in egg yolks, on the chemistry of iron during the in vitro enzymatic digestion of pinto beans. Three basic types of information were obtained. First, the total soluble iron was determined during in vitro enzymatic digestion under simulated oral, gastric (pH 2) and duodenal (pH 6) conditions. Phosvitin was found to have a strong solubilizing effect at pH 6 and pH 2 when in the presence of ascorbate. Pyrophosphate also leads to high iron mobilization. A second approach is to determine the static Fe2+ and Fe3+ concentrations following in vitro enzymatic digestion of pinto beans at pH 2 and pH 6. Ascorbic acid enhanced the total soluble iron at both pH values, however, only at pH 2 was a large proportion of the iron found in the Fe2+ state and then only in the presence of phosvitin but not pyrophosphate. A third approach is to determine the amount of Fe2+ formed in the digestive supernatant during a 10-min incubation with ferrozine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of iron and iron-complexes onKlebsiella pneumoniae (Klebsiella aerogenes)

Iron in the Fe(III) oxidation state had a negligible effect on the growth ofKlebsiella pneumoniae even at the highest concentration (0.45mm) obtainable without precipitation in a minimal medium containing glucose and inorganic salts together with Tris as the buffer and glycerol 2-phosphate as the phosphorus source. Nevertheless in its presence the toxic action of Cd²⁺, Zn²⁺ and Cu²⁺ was antagonized while that of Co²⁺ and Ni²⁺ was potentiated. Higher iron levels were obtained by supplementing the minimal medium with fructose, glycine, gluconate, tartrate and citrate at a range of concentrations. With fructose and glycine all of the resulting solutions were red-brown and non-toxic. This was also found with the other complexing agents when the ligand:iron ratios were low, but at higher ligand:iron ratios the solutions were green and toxic. Iron-citrate systems were especially toxic but resistance developed and was of the graded type. The results are discussed with particular reference to earlier physico-chemical studies by other workers and it is concluded that the red-brown colour is characteristic of the presence of polymers of high molar mass and that the green colour signifies the formation of low molar mass species.