Mobilization of iron from ferritin: new steps and details.

Much evidence indicates that iron stored in ferritin is mobilized through protein degradation in lysosomes, but concerns about this process have lingered, and the mechanistic details of its aspects are lacking. In the studies presented here, 59Fe-labeled ferritin was induced by preloading hepatic (HepG2) cells with radiolabeled Fe. Placing these cells in a medium containing desferrioxamine resulted in the loss of ferritin-59Fe, but adding high concentrations of reducing agents or modulating the internal GSH concentration failed to alter the rates of ferritin-59Fe release. Confocal microscopy showed that Fe deprivation increased the movement of ferritin into lysosomes and hyperaccumulation was observed when lysosomal proteolysis was inhibited. It also resulted in the rapid movement of DMT1 to lysosomes, which was inhibited by bafilomycin. Ferrihydrite crystals isolated from purified rat liver/spleen ferritin were solubilized at pH 5 and 7 by GSH, ascorbate, citrate and lysosomal fluids obtained from livers and J774a.1 macrophages. The inhibition of DMT1/Nramp2 and siRNA knockdown of Nramp1 each reduced the transfer of 59Fe from lysosomes to the cytosol; and hepatocyte-specific knockout of DMT1 in mice prevented the release of Fe from the liver responding to EPO treatment, but did not inhibit lysosomal ferritin degradation. We conclude that ferritin-Fe mobilization does not occur through changes in cellular concentrations of reducing/chelating agents but by the coordinated movement of ferritin and DMT1 to lysosomes, where the ferrihydrite crystals exposed by ferritin degradation dissolve in the lysosomal fluid, and the reduced iron is transported back to the cytosol via DMT1 in hepatocytes, and by both DMT1 and Nramp1 in macrophages, prior to release into the blood or storage in ferritin.

High dietary iron reduces transporters involved in iron and manganese metabolism and increases intestinal permeability in calves.

A 56-d experiment was designed to examine the effect of high dietary Fe on metal transporters involved in Fe and Mn metabolism. Fourteen weaned Holstein calves were stratified by weight and randomly assigned to 1 of 2 treatments: 1) no supplemental Fe (normal Fe) or 2) 750mg of supplemental Fe/kg of dry matter (high Fe). Jugular blood was collected on d 0, 35, and 56. At the end of the trial, 6 calves per treatment were humanely killed and duodenal scrapings, liver, and heart were collected for analysis. Additionally, proximal duodenum was mounted on Ussing chambers to assess intestinal barrier integrity. Calves receiving high dietary Fe displayed decreased transepithelial resistance and increased apical-to-basolateral flux of radiolabeled mannitol, suggesting that high Fe created increased intestinal permeability. Feeding calves a diet high in Fe decreased average daily gain, dry matter intake, and feed efficiency. Hemoglobin and serum Fe concentrations did not differ due to dietary treatment. High dietary Fe increased concentrations of Fe in the liver, but did not affect heart or duodenal Fe concentrations. Duodenal Mn concentrations were lowered by feeding a high Fe diet, but liver and heart Mn concentrations were not affected. As determined by real-time reverse transcription PCR, relative hepatic expression of the gene that encodes the Fe regulatory hormone hepcidin was 5-fold greater in calves fed high dietary Fe. Hepcidin is released in response to increased Fe status and binds to the Fe export protein ferroportin causing ferroportin to be degraded, thereby reducing dietary Fe absorption. Confirmation of this result was achieved through Western blotting of duodenal protein, which revealed that ferroportin was decreased in calves fed high dietary Fe. Duodenal protein expression of divalent metal transporter 1 (DMT1), a Fe import protein that can also transport Mn, tended to be reduced by high dietary Fe. Transcript levels of several genes involved in Fe metabolism in liver and duodenum were unchanged by treatment. In summary, feeding calves a diet high in Fe induced a signal cascade (hepcidin) designed to reduce absorption of Fe (via reduced protein expression of ferroportin and DMT1) in a manner similar to that reported in rodents. Additionally, reduced levels of DMT1 protein appeared to decrease duodenal Mn, suggesting that Mn may also be a substrate for DMT1 in cattle.

Technical note: copper chaperone for copper, zinc superoxide dismutase: a potential biomarker for copper status in cattle.

Copper chaperone for Cu, Zn superoxide dismutase (CCS) has been shown to be reflective of Cu status in mice and rats. The objective of this study was to evaluate liver and erythrocyte CCS as an indicator of Cu status in beef cattle (Exp. 1), and to test the acute-phase properties of CCS under conditions of inflammation (Exp. 2). In Exp. 1, samples of whole blood and liver were collected at slaughter (492 d of age) from 15 Cu-deficient and 6 Cu-adequate Angus calves. At the time of tissue collection, severe Cu deficiency had been achieved and differences (P < 0.0001) in plasma and liver Cu among Cu-adequate and Cu-deficient calves were extreme (1.26 vs. 0.19 mg/L and 208.4 vs. 6.3 mg/kg for plasma and liver Cu, respectively). Protein levels of CCS were greater in liver (40%; P = 0.02) and erythrocytes (65%; P < 0.0001) of Cu-deficient vs. Cu-adequate calves. In Exp. 2, inflammatory responses were elicited in beef heifers by administration of a Mannheimia hemolytica vaccine. Four days after vaccination, plasma concentrations of the Cu-dependent protein ceruloplasmin and the Cu-independent protein haptoglobin were increased (P < 0.001) by 71 and 83%, respectively. In contrast, detection of CCS protein in samples of liver and erythrocytes did not differ (P >or= 0.45) between baseline (d 0) and d 4 after vaccination. These data demonstrate that bovine erythrocyte and liver CCS protein levels increase in Cu-deficient cattle. Furthermore, levels of CCS protein do not change after a vaccine-induced inflammatory response, suggesting that unlike ceruloplasmin, CCS may be a reliable indicator of Cu status in cattle.

Expression of stimulator of Fe transport is not enhanced in Hfe knockout mice.

Hfe knockout (-/-) mice recapitulate many of the biochemical abnormalities of hereditary hemochromatosis (HH), but the molecular mechanisms involved in the etiology of iron overload in HH remain poorly understood. It was found previously that livers of patients with HH contained 5-fold higher SFT (stimulator of Fe transport) mRNA levels relative to subjects without HH. Because this observation suggests a possible role for SFT in HH, we investigated SFT mRNA expression in Hfe(-/-) mice. The 4- and 10-wk-old Hfe(-/-) mice do not have elevated levels of hepatic SFT transcripts relative to age-matched Hfe(+/+) mice, despite having 2.2- and 3.3-fold greater hepatic nonheme iron concentrations, respectively. Northern blot analyses of various mouse tissues revealed that SFT is widely expressed. The novel observation that SFT transcripts are abundant in brain prompted a comparison of SFT transcript levels and nonheme iron levels in the brains of Hfe(+/+) and Hfe(-/-) mice. Neither SFT mRNA levels nor nonheme iron levels differed between groups. Further comparisons of Hfe(-/-) and Hfe(+/+) mouse tissues revealed no significant differences in SFT mRNA levels in duodenum, the site of increased iron absorption in HH. Important distinctions between Hfe(-/-) mice and HH patients include not only differences in the relative rate and magnitude of iron loading but also the lack of fibrosis and phlebotomy treatment in the knockout animals.

Both iron deficiency and daily iron supplements increase lipid peroxidation in rats.

Numerous studies have shown that iron-loaded diets increase markers of lipid peroxidation in rats, but few have addressed the effects of oral iron supplements on these markers. We investigated the effects of daily and intermittent iron supplements on iron and vitamin E status, and lipid peroxidation. Iron supplements were administered in doses equivalent to those often given to pregnant women in the developing world. In Study 1, iron-deficient (D) and iron-normal (N) rats were fed either 0 or 8000 microgram of supplemental iron daily for 21 d. In Study 2, D rats were fed either the same supplements daily or once every 3 d (8 supplements total). Lipid peroxidation was assessed by breath ethane and pentane and by malondialdehyde (MDA) (using GC-MS). In Study 1, daily supplemented N and D rats had liver nonheme iron concentrations that were 1.8- and 2.7-fold higher, respectively, than those in unsupplemented N rats. Breath ethane levels were also higher in supplemented rats (P < 0.05), but MDA (in plasma, liver, kidney) and liver vitamin E did not differ. Unexpectedly, severely D, anemic rats had significant elevations in the levels of breath ethane, liver MDA and kidney MDA. In Study 2, liver iron and breath ethane decreased progressively (P < 0.05) from 1 d to 3 d after the last iron dose in intermittently supplemented rats. We conclude that iron deficiency results in lipid peroxidation, but that its correction with daily iron supplements results in abnormal iron accumulation and increased lipid peroxidation in rats. These effects are mitigated by intermittent iron supplementation.

Methods for measuring ethane and pentane in expired air from rats and humans.

Numerous studies in animals and humans provide evidence that ethane and pentane in expired air are useful markers of in vivo lipid peroxidation. The measurement of breath hydrocarbons, being noninvasive, is well suited for routine use in research and clinical settings. However, the lack of standardized methods for collecting, processing, and analyzing expired air has resulted in the use of a wide variety of different methods that have yielded highly disparate results among investigators. This review outlines the methods that we have developed and validated for measuring ethane and pentane in expired air from rats and humans. We describe the advantages of these methods, their performance, as well as potential errors that can be introduced during sample collection, concentration, and analysis. A main source of error involves contamination with ambient-air ethane and pentane, the concentrations of which are usually much greater and more variable than those in expired air. Thus, it appears that the effective removal of ambient-air hydrocarbons from the subject's lungs before collection is an important step in standardizing the collection procedure. Also discussed is whether ethane or pentane is a better marker of in vivo lipid peroxidation.

A practical and reliable method for measuring ethane and pentane in expired air from humans.

We describe a method for the collection of expired air and further document the performance of our analytical technique that is used to measure ethane and pentane simultaneously. Four minutes of breathing hydrocarbon-free air before collection effectively removed high concentrations of residual ambient ethane and pentane from the lungs, with washout times up to 30 min resulting in no further reductions in breath hydrocarbons. Mean (+/-SE) exhalation rates (pmol/kg b.wt./min) in 11 subjects were 2.4 +/- 0.6 for ethane and 1.5 +/- 1.3 for pentane. Total intraindividual variability in exhalation rates (as percent coefficient of variation, %CV), measured from 4 subjects on at least 6 different days, was greater for pentane (44% CV) than for ethane (29% CV). Analytical variability contributed 6% to the total %CV. Advantages of the method are described, and reasons for the large variability in values reported in the literature are discussed.

Concentrating breath samples using liquid nitrogen: a reliable method for the simultaneous determination of ethane and pentane.

The measurement of ethane and pentane in breath offers a sensitive and noninvasive means to assess in vivo lipid peroxidation in animals and humans. However, numerous technical obstacles inherent in collecting and concentrating air-breath samples have limited the wider application of these measurements for the assessment of in vivo lipid peroxidation. We have developed a relatively simple, inexpensive, rapid, and reliable method to collect, concentrate, and measure breath ethane and total-body pentane from rats. This method, which concentrates alkanes from 4 liters of collected air-breath on adsorbant cooled to -174 degrees C, was found to be superior to similar cryofocusing techniques at -130 degrees C, which fail to effectively trap highly volatile ethane from large volumes of air. We found ethane evolves predominantly through breath, whereas a significant amount of pentane evolves from sources other than breath. Mean evolution rate for ethane was 1.08 pmol/100 g body wt/min. Pentane evolution rates displayed more inter-rat and day-to-day variability with a mean of 0.52 pmol/100 g body wt/min. We also found that excreted rat feces exude large amounts of ethane and pentane.