Secreted pyomelanin of Legionella pneumophila promotes bacterial iron uptake and growth under iron-limiting conditions.

Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

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 ((59)Fe and (55)Fe) 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.

Zinc inhibits nonheme iron bioavailability in humans.

There is increasing concern about potential negative interactions in combined iron and zinc supplementation. The aim of the present study was to determine the dose-response effect of zinc, given as a solution, on iron bioavailability. Twenty-two healthy adult women were selected to participate in the study. Iron, with or without zinc was given as an aqueous solution on d 1,2,14, and 15 of the study. Iron bioavailability was measured on the basis of erythrocyte incorporation of 55Fe or 59Fe 14 d after administration. Subjects received 0.5 mg of iron together with graded zinc concentrations (0-11.71 mg). No significant effect of zinc on iron absorption was found at Zn:Fe molar ratios up to 2:1. At 5:1,10:1, and 20:1 molar ratios, a dose-dependent inhibitory effect on iron absorption was observed (28-40% of iron absorption inhibition; one-way repeated-measures ANOVA, F=4.48, p=0.02). In conclusion, zinc administration combined with iron in an aqueous solution leads to the inhibition of iron bioavailability, which occurs in a dose-dependent way. This negative interaction should be considered for supplementation programs with both microminerals.

Ascorbyl palmitate enhances iron bioavailability in iron-fortified bread.

BACKGROUND: One of the strategies to control iron deficiency anemia is the fortification of food with iron. A mechanism for improving the bioavailability of iron is to add an iron absorption promoter. OBJECTIVE: The objective was to determine the effect of ascorbyl palmitate (AP) on the bioavailability of iron in fortified bread made from refined wheat flour. DESIGN: The iron bioavailability of wheat flour fortified with either ferrous sulfate alone or ferrous sulfate plus AP was studied with the use of double radio iron (55Fe and 59Fe) erythrocyte incorporation in 14 women. RESULTS: Geometric mean (+/- range of 1 SD) iron absorption from the bread fortified with ferrous sulfate was 10.5% (4.1-27.0%). The addition of AP at molar ratios of AP to Fe of 2:1 and 4:1 significantly increased iron absorption [14.6% (5.9-36.1%) and 20.2% (10.6-38.6%), respectively; P < 0.001]. CONCLUSION: AP is a strong promoter of iron absorption from fortified bread because of its thermoresistant properties.

Nutritional and technological behavior of stabilized iron-gluconate in wheat flour.

Food fortification has been shown to be an effective strategy to overcome iron malnutrition. When a new iron compound is developed for this purpose, it must be evaluated from a nutritional and technological point of view before adding it into foods. In this way, we have evaluated ferrous gluconate stabilized by glycine as a new iron source to be used in wheat flour fortification. We performed biological studies in rats as well as sensory perceptions by human subjects in wheat flour fortified with this iron source. The productions of pentane as a rancidity indicator as well as the change of the sensorial properties of the biscuits made with stabilized ferrous gluconate-fortified wheat flour were negligible. Iron absorption in water from this iron source was similar to the reference standard ferrous sulfate. Nevertheless, because of the phytic acid content, iron absorption from fortified wheat flour decrease 40% for both iron sources. The addition of zinc from different sources did not modify iron absorption from ferrous sulfate and stabilized ferrous gluconate in water and wheat flour. The iron absorption mechanism as well as the biodistribution studies demonstrate that the biological behavior of this iron source does not differ significantly from the reference standard. These results demonstrate that the iron source under study has adequate properties to be used in wheat flour fortification. Nevertheless, more research is needed before considering this iron source for its massive use in food fortification.

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells.

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.

Alteration in the organ distribution of iron by truncated transferrin: implications for iron chelation therapy.

The ability of the partial molecule of transferrin, truncated transferrin (t-Tf), to act as an excretable biologic iron chelator was examined. We confirmed the observations of Zak and Aisen (Zak O, Aisen P. Biochem Biophys Acta 1985;1952:24-8) that thermolysin treatment of human transferrin produces half molecules that retain iron-binding capacity. These molecules are poorly recognized by surface receptors on either human or murine cells. Although the plasma half-life of human transferrin in mice is moderately long (40 hours), injection of t-Tf into mice results in its rapid clearance (half-life = 10 minutes). Injection of iron 59-labeled transferrin results in the deposition of iron in the major hematopoetic organs of mice such as the spleen, bone marrow, and liver. Injection of 59Fe-labeled t-Tf results in the quantitative recovery of iron in the kidneys: 59Fe is retained in the kidney for substantial periods of time with little evidence of its excretion into urine. Injection of iodine 125-labeled t-Tf also results in the deposition of radioactivity in the kidneys, but 125I is rapidly excreted into the urine, where it is detected as free iodine. These results indicate that although t-Tf is directed to the kidney and filtered by the glomerulus, the molecule is reabsorbed and degraded, and iron is retained. These results have implications in the design of iron chelators.

Blood flow to bone marrow during development of anemia or polycythemia in the rat.

We applied the radioactive microsphere method to follow the magnitude and time course (0 to 96 hours) of blood flow changes during development and recovery from anemia in awake rats. Blood flow was also monitored during a 96-hour period after polycythemia was induced (erythropoietin administered subcutaneously [SC]). The possible influence of innervation was also examined. After a blood loss of approximately 50% (hypovolemia), blood flow to the femoral marrow tripled within 12 hours and remained elevated for the entire 96-hour period. The relative increase in blood flow to the femoral bone was even greater. Similar findings were obtained in rats with phenylhydrazine (PHZ) hemolytic anemia (normovolemia). Denervation had no detectable effect on the increased blood flow to either marrow or bone. The augmented blood flow during hemolytic anemia was accompanied by a doubling of the oxygen consumption rate by the marrow, while the glucose uptake was not detectably altered. Erythropoietin supplements (3 x 1,000 IU/kg, SC, 6-hour intervals) increased blood flow to the marrow by approximately 25% after 48 hours, and at 72 hours the blood flow had reached a value twice that obtained under control conditions. These results indicate that blood flow to bone marrow is highly variable and hormonally and/or locally regulated. This may have practical consequences for marrow transplantation technology and for administration of drug therapy to patients with insufficient bone marrow hematopoiesis.

Effects of dietary iron deficiency and tungsten supplementation on 59Fe absorption and gastric retention from 59Fe compounds in rats.

1. In vivo 59Fe absorption from intrinsically labelled Fe-containing fractions of liver and blood were measured in rats by intragastric dosing. All rats were fed on a low-Fe diet for 3 d before dosing in order to standardize the Fe status of the intestinal mucosal cells. 2. An increase in digestion time from 2 to 12 h increased 59Fe absorption (P less than 0.01) from all fractions except ferritin. 3. Fe-deficient rats when compared with essentially Fe-replete rats showed decreased gastric retention for all fractions, but increased 59Fe absorption over 2 h only from ferritin. Ferritin showed several unusual absorption characteristics. 4. Dietary tungsten supplementation of Fe-deficient rats reduced the ferroxidase activity of intestinal mucosal xanthine oxidase. In addition, gastric retention and 59Fe absorption (P less than 0.05) from all fractions were increased.

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole.

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.

Judgement of measured values of intestinal iron absorption.

The value of a medicinal iron preparation is influenced by several factors e.g. therapeutical efficacy, frequency of side effects, and prescriptions for intake. Since the accurate quantitation of therapeutical efficacy is difficult to establish, the value of fractional intestinal absorption of iron, which is a prerequisite for the effect on haemoglobin regeneration, represents a useful criterion for that particular factor. When intestinal absorption is investigated the selection of subjects is of importance. Studies should be performed on subjects with iron deficiency since the adaptation of intestinal absorption to the body's need is the basis of oral iron supplementation. Different methods have been used to assess intestinal absorption, some of which give almost identical results or show a close correlation. However, mixing data from different investigations in order to convert semi-quantitative values obtained from plasma iron tolerance curves into amounts of absorbed iron by means of regression equations estimated from studies on different subjects and under different conditions may lead to considerable uncertainties of the calculated results. Even more difficult to evaluate is the relation between fractional absorption and costs of therapy, since there are additional factors influencing the duration of supplementation and therefore the total amount of iron necessary.

Myelotoxicity and macrophage alteration in mice exposed to ochratoxin A.

Six- to seven-week-old female B6C3F1 mice were administered a total of 0, 20, 40, or 80 mg/kg of ochratoxin A (OCT A) ip on alternate days over an 8-day period. Twenty-four hours following the final dose, histopathology, bone marrow, and macrophage parameters were assayed. There was a dramatic dose related decrease in thymic mass with the mean thymus weight of the high dose animals being only 33% of controls. Histologic evidence of nephrotoxicity was minimal and restricted to the inner cortex. Myelotoxicity was present as evidenced by bone marrow hypocellularity, decreased marrow pluripotent stem cells (CFU-S), granulocyte-macrophage progenitors (CFU-GMs), and decreased 59Fe uptake in marrows and spleens of exposed mice. Peritoneal macrophages from sc as well as ip injected mice demonstrated increased phagocytic capacities and increased capacity to inhibit tumor cell growth. These alterations in bone marrow cells and macrophages suggest myelotoxicity is an additional potential hazard of OCT A exposure.

Evaluation of iron-chelating agents in cultured heart muscle cells. Identification of a potential drug for chelation therapy.

Primary cultures of neonatal rat cardiac muscle cells incorporated radioiron from both [55Fe]transferrin and 59FeCl3 (added simultaneously). To evaluate the effect of iron chelators on such uptake, deferri chelators were added 6 hr after addition of the radioiron sources. The microbial chelator agrobactin was significantly more effective than the drug defoxamine in reduction of 55Fe uptake from [55Fe]transferrin; both chelators halted 59Fe3+ uptake. Agrobactin may have potential in chelation therpay for iron-overload disease. Certain other microbial chelators lowered radioiron uptake from either [55Fe]transferrin of 59FeCl3. These chelators should be useful inhibitors for studies of animal cell iron uptake and intracellular iron flow.

Iron sufficiency in breast-fed infants and the availability of iron from human milk.

Four infants were studied who had been exclusively breast-fed for periods varying from 8 to 18 months. All had grown sufficiently to have exhausted their prenatally acquired iron endowment with respect to meeting current needs for maintaining normal hemoglobin levels. All infants had normal hemoglobin values and normal serum iron values. Studies of iron absorption from breast milk and cow's milk were performed in ten normal adults. The absorption of iron from the human milk was significantly higher. These findings suggest that the iron present in human milk is sufficient to meet the iron requirements of the exclusively breast-fed infant until he approximately triples his birthweight.