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• W4308295085 abstract "The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(−/−) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(−/−) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions. Liquid chromatography detected by inductively coupled plasma mass spectrometry of flow-through solutions exhibited low-molecular-mass iron peaks that did not increase intensity with increasing dietary iron. Retentates yielded peaks due to transferrin (TFN) and ferritin, but much iron in these samples adsorbed onto the column. Retentates treated with the chelator deferoxamine (DFO) yielded a peak that comigrated with the Fe–DFO complex and originated from iron that adhered to the column in the absence of DFO. Additionally, plasma from younger and older 57Fe-enriched HFE mice were separately pooled and concentrated by ultrafiltration. After removing contributions from contaminating blood and TFN, Mössbauer spectra were dominated by features due to magnetically interacting FeIII aggregates, with greater intensity in the spectrum from the older mice. Similar features were generated by adding 57FeIII to “pseudo plasma”. Aggregation was unaffected by albumin or citrate at physiological concentrations, but DFO or high citrate concentrations converted aggregated FeIII into high-spin FeIII complexes. FeIII aggregates were retained by the cutoff membrane and adhered to the column, similar to the behavior of NTBI. A model is proposed in which FeII entering blood is oxidized, and if apo-TFN is unavailable, the resulting FeIII ions coalesce into FeIII aggregates, a.k.a. NTBI. The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(−/−) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(−/−) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions. Liquid chromatography detected by inductively coupled plasma mass spectrometry of flow-through solutions exhibited low-molecular-mass iron peaks that did not increase intensity with increasing dietary iron. Retentates yielded peaks due to transferrin (TFN) and ferritin, but much iron in these samples adsorbed onto the column. Retentates treated with the chelator deferoxamine (DFO) yielded a peak that comigrated with the Fe–DFO complex and originated from iron that adhered to the column in the absence of DFO. Additionally, plasma from younger and older 57Fe-enriched HFE mice were separately pooled and concentrated by ultrafiltration. After removing contributions from contaminating blood and TFN, Mössbauer spectra were dominated by features due to magnetically interacting FeIII aggregates, with greater intensity in the spectrum from the older mice. Similar features were generated by adding 57FeIII to “pseudo plasma”. Aggregation was unaffected by albumin or citrate at physiological concentrations, but DFO or high citrate concentrations converted aggregated FeIII into high-spin FeIII complexes. FeIII aggregates were retained by the cutoff membrane and adhered to the column, similar to the behavior of NTBI. A model is proposed in which FeII entering blood is oxidized, and if apo-TFN is unavailable, the resulting FeIII ions coalesce into FeIII aggregates, a.k.a. NTBI. Nontransferrin-bound iron (NTBI) is a toxic form of iron in the blood of individuals with iron-overload diseases such as hereditary hemochromatosis (1Anderson G.J. Bardou-Jacquet E. Revisiting hemochromatosis: genetic vs. phenotypic manifestations.Ann. Transl. Med. 2021; 9: 731Crossref PubMed Google Scholar) and β-thalassemia (2Chauhan W. Shoaib S. Fatma R. Zaka-ur-Rab Z. Afzal M. Beta-thalassemia and the advent of new interventions beyond transfusion and iron chelation.Br. J. Clin. Pharmacol. 2022; 88: 3610-3626Crossref PubMed Scopus (5) Google Scholar). It accumulates excessively in the liver and other organs, resulting in liver fibrosis, cirrhosis, cancer, endocrinopathies, and cardiomyopathies (3Knutson M.D. Non-transferrin-bound iron transporters.Free Rad. Biol. Med. 2019; 133: 101-111Crossref PubMed Scopus (97) Google Scholar). Treatments include low iron diets, frequent phlebotomies, and ingestion of iron-binding chelators. Effectiveness is limited, especially for β-thalassemia. Transferrin (TFN) is an iron-binding protein in the blood which serves as an iron buffer (4Bartnikas T.B. Known and potential roles of transferrin in iron biology.Biometals. 2012; 25: 677-686Crossref PubMed Scopus (37) Google Scholar). In healthy individuals, about one-third of TFN is in the holo- (two FeIII ions bound) form whereas most of the remainder is apo- (iron-free). Nutrient iron enters the blood via ferroportin (FPN), a membrane-bound FeII transporter that is highly expressed on the basolateral side of enterocytes in the duodenum (5Ganz T. Hepcidin and iron regulation, 10 years later.Blood. 2011; 117: 4425-4433Crossref PubMed Scopus (710) Google Scholar). Holo-TFN is distributed throughout the body and enters cells via TFN receptor–mediated endocytosis. FPN is also highly expressed in macrophages, including Kupffer cells in the liver and red-pulp macrophages in the spleen. In both cases, FPN functions to release stored iron into the blood. Iron import into the body is regulated at the systems’ level by hepcidin, a small peptide hormone produced in the liver in response to excessive bodily iron (5Ganz T. Hepcidin and iron regulation, 10 years later.Blood. 2011; 117: 4425-4433Crossref PubMed Scopus (710) Google Scholar). Hepcidin binds FPN, causing its internalization and subsequent hydrolytic degradation. Individuals with hemochromatosis, most commonly harboring a mutation in the HFE (Homeostatic Fe regulator) gene, generate insufficient hepcidin, resulting in excessive levels of FPN and thus excessive import of nutrient iron into the blood. This saturates TFN such that the concentration of apo-TFN available to receive newly imported iron is insufficient. The excessive iron released into the blood becomes NTBI. Despite being recognized to exist for a half-century, the chemical identity of NTBI remains unestablished (1Anderson G.J. Bardou-Jacquet E. Revisiting hemochromatosis: genetic vs. phenotypic manifestations.Ann. Transl. Med. 2021; 9: 731Crossref PubMed Google Scholar, 2Chauhan W. Shoaib S. Fatma R. Zaka-ur-Rab Z. Afzal M. Beta-thalassemia and the advent of new interventions beyond transfusion and iron chelation.Br. J. Clin. Pharmacol. 2022; 88: 3610-3626Crossref PubMed Scopus (5) Google Scholar, 3Knutson M.D. Non-transferrin-bound iron transporters.Free Rad. Biol. Med. 2019; 133: 101-111Crossref PubMed Scopus (97) Google Scholar, 4Bartnikas T.B. Known and potential roles of transferrin in iron biology.Biometals. 2012; 25: 677-686Crossref PubMed Scopus (37) Google Scholar, 5Ganz T. Hepcidin and iron regulation, 10 years later.Blood. 2011; 117: 4425-4433Crossref PubMed Scopus (710) Google Scholar, 6Faber M. Jordal R. Presence of 2 iron-transport proteins in serum.Nature. 1961; 192: 181Crossref PubMed Scopus (2) Google Scholar, 7Sarkar B. State of iron(III) in normal human serum: low molecular weight and protein ligands besides transferrin.Can. J. Biochem. 1970; 48: 1339-1350Crossref PubMed Scopus (60) Google Scholar, 8Hershko C. Graham G. Bates G.W. Rachmilewitz E.A. Nonspecific serum iron in thalassemia: an abnormal serum iron fraction of potential toxicity.Br. J. Haematol. 1978; 50: 255-263Crossref Scopus (379) Google Scholar, 9Graham G. Bates G.W. Rachmilwitz E.A. Hershko C. Nonspecific serum iron in Thalassemia – quantitative and chemical reactivity.Am. J. Hematol. 1979; 6: 207-217Crossref PubMed Scopus (46) Google Scholar, 10Batey R.G. Fracp P. Fong L.C. Shamir S. Sherlock S. A non-transferrin-bound serum iron in idiopathic hemochromatosis.Dig. Dis. Sci. 1980; 25: 340-346Crossref PubMed Scopus (144) Google Scholar, 11Evans R.W. Rafique R. Zarea A. Rapisarda C. Cammack R. Evans P.J. et al.Nature of non-transferrinbound iron: studies on iron citrate complexes and thalassemic sera.J. Biol. Inorg. Chem. 2008; 13: 57-74Crossref PubMed Scopus (128) Google Scholar, 12Silva A.M. Kong X. Parkin M.C. Cammack R. Hider R.C. Iron(III) citrate speciation in aqueous solution.Dalton Trans. 2009; 40: 8616-8625Crossref PubMed Scopus (187) Google Scholar, 13Brissot Pierre Loreal Olivier Iron metabolism and related genetic diseases: a cleared land, keeping mysteries.J. Hepatol. 2016; 64: 505-515Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Silva A.M.N. Rangel M. The (Bio)Chemistry of non-transferrin-bound iron.Molecules. 2022; 27: 1784Crossref PubMed Scopus (9) Google Scholar). One reason for this is that its two most predominant characteristics—accumulating in organs and susceptibility to chelation—are indirect and difficult to quantify. Iron accumulates in organs primarily as ferritin (FTN), but NTBI is a different species that is likely altered via reduction and ligand exchange as it enters the cell and is converted into FTN. NTBI is typically defined operationally as the iron in plasma that reacts with a particular chelator under prescribed concentrations and durations. However, the size of the NTBI pool in plasma is affected by these details. A second problem is that the concentration of NTBI in diseased plasma is not exceptionally high (1–10 μM), and although NTBI concentration in healthy individuals is lower, it is still detectable and significant. A third problem arises from the gradual and ambiguous “spillover” conditions required to generate NTBI; between 40% and 70% TFN saturation is reportedly sufficient for NTBI levels to increase (10Batey R.G. Fracp P. Fong L.C. Shamir S. Sherlock S. A non-transferrin-bound serum iron in idiopathic hemochromatosis.Dig. Dis. Sci. 1980; 25: 340-346Crossref PubMed Scopus (144) Google Scholar, 15Aruoma O.I. Bomford A. Polson R.J. Halliwell B. Nontransferrin-bound iron in plasma from hemochromatosis patients – effect of phlebotomy therapy.Blood. 1988; 72: 1416-1419Crossref PubMed Google Scholar, 16Breuer W. Ronson A. Slotki I.N. Hershko C. Cabantchik Z.I. The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation.Blood. 2000; 95: 2975-2982Crossref PubMed Google Scholar). A fourth problem is that the aqueous redox and coordination chemistry of iron is complicated, and NTBI may be heterogeneous (12Silva A.M. Kong X. Parkin M.C. Cammack R. Hider R.C. Iron(III) citrate speciation in aqueous solution.Dalton Trans. 2009; 40: 8616-8625Crossref PubMed Scopus (187) Google Scholar, 17Breuer W. Hershko C. Cabantchik Z.I. The importance of non-transferrin bound iron in disorders of iron metabolism.Transfus. Sci. 2000; 23: 185-192Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Two basic types of experiments have contributed to our understanding of NTBI, but perhaps they have also hindered it. One type of experiment has been to monitor the fate of added radioactive 59Fe to sera or plasma. Radioactive iron binds preferentially and tightly to available apo-TFN, and the excess is concluded to be (or become) NTBI. This assumes that the only iron-binding ligand in sera/plasma is the NTBI ligand (symbolized :LNTBI) and that this ligand is present in excess. Actually, there are many potential ligands in plasma (18May P.M. Linder P.W. Computer simulation of metal-ion equilibria in biofluids: models for the low-molecular-weight complex distribution of Calcium(II), Magnesium(II), Manganese(II), Iron(III), Copper(II), Zinc(II) and Lead(II) ions in human blood plasma.J. Chem. Soc., Dalton Trans. 1977; : 588-595Crossref Google Scholar) and the added 59Fe may bind any or all of them. In the other type of experiment, a chelator is added to sera/plasma, and the resulting Fe–chelator complex is assumed to arise from the binding of NTBI; the assumed general reaction is {Fe:LNTBI + chelator ⇄ Fe-chelator +:LNTBI}. The problem is that NTBI is destroyed during this reaction, making it unlikely that such experiments can ultimately be used to identify NTBI. Moreover, the added chelator might also sequester iron that is bound to other non-NTBI species, overestimating the size of the NTBI pool. To avoid these problems, we and others (19Neu H.M. Alexishin S.A. Brandis J.E.P. Williams A.M.C. Li W.J. Sun D.J. et al.Snapshots of iron speciation: tracking the fate of iron nanoparticle drugs via a liquid chromatography-inductively coupled plasma-mass spectrometric approach.Mol. Pharm. 2019; 16: 1272-1281Crossref PubMed Scopus (11) Google Scholar) have investigated untreated blood using liquid chromatography (LC) interfaced to an online inductively coupled plasma mass spectrometer (LC-ICP-MS), and have detected and characterized endogenous iron-containing complexes without adding iron or chelators. Neu et al. (19Neu H.M. Alexishin S.A. Brandis J.E.P. Williams A.M.C. Li W.J. Sun D.J. et al.Snapshots of iron speciation: tracking the fate of iron nanoparticle drugs via a liquid chromatography-inductively coupled plasma-mass spectrometric approach.Mol. Pharm. 2019; 16: 1272-1281Crossref PubMed Scopus (11) Google Scholar) detected FeIII(citrate) in human blood plasma by ESI-MS, supporting the conclusion that NTBI = FeIII citrate. Dzubia et al. (20Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron in healthy blood plasma is not predominately ferric citrate.Metallomics. 2018; 10: 802-817Crossref PubMed Google Scholar) detected low concentrations of low-molecular-mass (LMM) iron species in plasma from healthy humans, horses, mice, and pigs, but the chromatographic properties of these species largely differed from those of FeIII(citrate). Unexpectedly, the 10 kDa plasma ultrafiltrate (or flow-through solution, FTS) from human hemochromatosis patients did not exhibit any additional iron-detected LC peaks relative to healthy controls. However, the patients had been treated for the disease, suggesting that their NTBI levels might have been too low to detect. Dzubia et al. (21Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.Metallomics. 2019; 11: 1900-1911Crossref PubMed Google Scholar) hypothesized that the low concentration of LMM iron species observed in the earlier study was due to the quantitative removal of NTBI by the liver, and so they surgically implanted catheters in the portal vein of pigs that had been starved for iron. Intestinal blood passes through this vein to the liver, and so they anticipated that blood removed from it would contain high concentrations of NTBI. Blood was also removed from the caudal/cranial vena cava as a control. A bolus of 57Fe was injected into the stomach via a feeding tube, and blood samples were removed from both catheters at increasing times. Since only 2% of natural-abundance iron is due to 57Fe, the fate of the injected enriched 57Fe could be followed. Surprisingly, the LMM iron complexes did not become enriched in 57Fe; rather the injected 57Fe bound apo-TFN. Dzubia et al. (21Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.Metallomics. 2019; 11: 1900-1911Crossref PubMed Google Scholar) concluded that the detected LMM iron complexes arise from internal stores rather than directly from nutrient iron. Building off those results, we hypothesized that NTBI might only be detectable using iron-overloaded genetically modified animals. Here, we investigated this by examining HFE(−/−) (heretofore called HFE) mice fed iron-deficient diets supplemented with 0, 50, 500, and 5000 mg of natural abundance FeIII citrate. We selected the HFE gene because a mutation in it is the most common origin of the disease (22Barton J.C. Edwards C.Q. Acton R.T. HFE gene: structure, function, mutations, and associated iron abnormalities.Gene. 2015; 574: 179-192Crossref PubMed Scopus (76) Google Scholar). Using our LC-ICP-MS system, we expected to observe increasing levels of LMM iron complexes in the blood plasma filtrates of these sick animals, especially as the concentration of iron in the diet increased. Once again, our results were unexpected, but they prompted a new and intriguing hypothesis as to the chemical nature of NTBI. Figure 1 outlines the chronology of our study. Our initial objective was to detect and characterize NTBI in the blood of HFE mice (and controls) raised on 0, 50, 500, and 5000 mg of iron per kg of iron-deficient chow. Some HFE mice were raised on 50 mg 57Fe/kg chow. Mice were sacrificed at various ages, blood was collected, and the plasma portion was filtered through a 10 kDa cut-off membrane, resulting in retentate and FTS fractions. A flow chart showing an overview of the study is given in Fig. S1. We initially focused on detecting NTBI in FTSs since we expected it to be a LMM species. FTSs of the plasma from both HFE and control mice were subjected to LC-ICP-MS chromatography using a size-exclusion low-mass column that could resolve species with masses ranging from 100 to 10,000 Da. In a typical experiment performed over the course of a day, eight HFE or control mice were sacrificed, two from each diet group. Adult mice yielded only 0.5 to 1 ml of blood, and so blood of two mice from each group (3 for younger mice) were combined. After centrifugation, plasma fractions, representing ∼ 50% of total volume, were collected and filtered, yielding ∼ 70 μl of retentate and ∼ 400 μl of FTS. The iron concentration in HFE FTS (from six 12–16 weeks mice) was modest, 1.9 ± 0.2 μM Fe. Plasma contains hundreds of mM of salt which fouled the ICP-MS instrument and reduced the detector response. Significant amounts of iron adsorbed onto the column, and subsequent cleaning had limited effectiveness. Small differences in daily tuning of the ICP-MS and changes in columns caused shifts in peak intensities and/or elution volumes. As a result, traces obtained on different days were not easily compared, so analyses were limited to assessing overall patterns within a group of traces obtained on the same day. Chromatograms of FTS from HFE and control mice exhibited 1 to 4 low-intensity iron peaks (Fig. 2). Results of three separate experiments are shown, including from 3-weeks HFE mice (Panel A), 12 weeks HFE mice (Panel B), and 16 weeks control mice (Panel C). Iron-detected traces from four additional experiments are shown in Fig. S2, including FTS from 3 weeks HFE (Panel B), 24 weeks HFE (Panel C), 32 weeks HFE (Panel A), and 24 weeks control (Panel D) mice. Specific peaks within a group were nearly identical in terms of elution volumes and intensities. Peaks in SI experiments were more intense; however, some of that intensity was due to contaminating iron that had desorbed from the column with each sample injection. In the experiment of Fig. S2D, we verified this by running a “ghost” column (i.e., peek tubing in place of the column) and measuring the area under the resulting iron peaks. Although this indicated substantial contaminating iron in LC traces, the nearly identical intensity of the ghost column peaks confirmed that the concentration of iron in the FTS did NOT increase proportionately with dietary iron. All such experiments indicated the same. In the experiment of Fig. S2C, an FeIII citrate standard run on the same day did not comigrate with the Fe peaks from plasma, supporting our previous conclusion (20Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron in healthy blood plasma is not predominately ferric citrate.Metallomics. 2018; 10: 802-817Crossref PubMed Google Scholar, 21Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.Metallomics. 2019; 11: 1900-1911Crossref PubMed Google Scholar) that FeIII citrate was not the dominant LMM Fe species in plasma FTS. The LMM iron species in the FTS of HFE (or control) mice were present at low concentrations and were similar regardless of whether the sample originated from HFE or control mice and regardless of the iron level in the diet. Overall, these results forced us to conclude that plasma FTS from HFE mice do not accumulate a LMM form of iron even on a high-iron diet. With some reluctance, we shifted the search for NTBI to retentate fractions. Such fractions from 24 weeks control and HFE mice were run on a high-mass column which resolved species with masses between 10 kDa and 100 kDa (Fig. 3, A and B). Matching ghost column traces (×0.1) are in red. Two major peaks were observed, which were assigned to FTN and TFN by running protein standards of horse spleen FTN (Sigma) and human TFN (Athens Research). The pattern in Fig. 3A suggested that the TFN peak intensity in HFE retentates increased from 0 to 50 mg Fe and then remained relatively constant. The peak assigned to FTN increased gradually with dietary iron. For control retentates, the FTN peak also increased with dietary iron (Fig. 3B). No such trend was evident for TFN. Other batches of retentate analyzed by LC-ICP-MS showed increasing TFN and FTN saturation with increasing Fe in the diet. Ghost column areas in Fig. S3A indicated little contamination; however, 35% to 70% of sample iron typically adsorbed on the column. Retentates from control and HFE mice were devoid of LMM species (Fig. S4). The intensity of the void volume peaks increased with increasing nutrient iron, especially for the HFE samples. NTBI is commonly quantified by its reaction with deferoxamine (DFO), and so we treated retentate samples with this chelator to assess the presence of NTBI in those fractions. Some HFE mice were raised on a “regular” diet containing ∼250 mg iron/kg chow. Retentate samples from 24 to 36 weeks “regular” mice (called R24 and R36) were diluted 80-fold and divided in halves. Half of each sample was treated with DFO, and half was left untreated. All four samples were run on the high-mass and ghost columns (Fig. 4). A trace of an Fe-DFO standard was also collected. The dominant Fe-detectable peak for both R24 and R36 mice originated from TFN, and its intensity was similar for all four traces. Traces from the two samples that had been treated with DFO exhibited a second major peak which comigrated with the Fe-DFO standard. The intensity of these peaks did not increase at the expense of the TFN peak, rather the peaks simply appeared. We concluded that HFE retentates contain a “sticky” form of iron that adsorbed onto the column but was also chelatable by DFO. We will ultimately conclude that this material is NTBI. The corresponding ghost column peaks indicated that the R36 retentate contained more than twice as much iron as the R24 retentate, suggesting that the DFO-chelatable iron increased with age, as expected for NTBI. Citrate is the most popular candidate for the NTBI ligand, and so we performed experiments to explore this possibility. We attempted to remove LMM species from plasma, including FeIII citrate, by filtering plasma from 36-weeks HFE “regular” mice using a 10 kDa cutoff membrane and washing the retentate twice with water. The retentate was then divided in two, half was treated with citrate, and half was untreated. Both halves were passed through the high-mass column. In both traces, the dominant peak was TFN (Fig. 5 Panel A). Quantification of the ghost-column trace indicated that 40% of the iron in the washed untreated retentate sample was not bound to TFN and was not detected in the trace. This undetected form of iron must have adsorbed to the column (ultimately, we will assign it to NTBI). The citrate-treated sample exhibited new peaks in the LMM region (at ∼ 22 ml) with approximately the intensity expected if citrate coordinated the high-mass “sticky” (NTBI) iron that was undetected in the other trace. We write this as {FeIII:LNTBI + citrate → FeIIIcitrate +:LNTBI}. Two similar experiments using plasma FTSs were analyzed using the low-mass column. In one experiment, FTSs from plasma of six mice were combined and then divided in two. Half was treated with an FeIII citrate solution (which contained an excess of citrate), and half was untreated. An FeIII citrate standard was also run. The trace of the untreated half exhibited ∼ 3 resolvable peaks at 16 to 21 ml elution volume plus a broad peak at longer elution volumes (Fig. 5B, middle trace). The trace of the treated sample (upper trace) was qualitatively similar but more intense. It included a peak that nearly comigrated with the FeIII citrate standard (perhaps the slight shift was due to the salt present only in the FTS sample), but the intensity of the “FeIII citrate” peak was modest relative to the increased intensities of the other peaks in the trace. (Note: the elution volume of FeIII citrate that migrated through the low-mass column differed from that through the high-mass column.) We concluded that most (∼90%) of the iron from the added FeIII citrate underwent ligand exchange with other species in the FTS. This suggested that FeIII citrate is not the most stable iron complex formed when citrate is added to plasma; much of it converts to other forms resulting in a distribution of species. If NTBI was present in retentate fractions rather than in FTSs, we realized that it might be possible to detect using Mössbauer spectroscopy (MB). With a limited amount of 57Fe-enriched plasma available from a recent study (23Vali S.W. Lindahl P.A. Mössbauer spectroscopic characterization of organs from HFE(-/-) mice: relevance to hereditary hemochromatosis.J. Biol. Chem. 2022; Google Scholar), we decided to combine plasma from the four youngest HFE mice that were available (4, 6, 10, and 14 weeks), concentrate the sample using a 10 kDa cutoff membrane, and load it into a MB cup for analysis. We did the same for plasma from four older HFE mice (18, 24, 32, and 52 weeks). Since no 57Fe-enriched plasma from control mice were available to serve as a control, we anticipated that NTBI might be present in greater amounts in the old mice sample. The iron concentrations in the young and old retentates (after removing residual red blood cell contributions) were 96 ± 6 μM and 136 ± 5 μM, respectively. Since the retentates were concentrated ∼ 5-fold for this experiment, we estimate that the plasma iron concentration would be ∼ 20 μM in young and ∼ 27 μM in old plasma, similar to our previous values (20Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron in healthy blood plasma is not predominately ferric citrate.Metallomics. 2018; 10: 802-817Crossref PubMed Google Scholar, 21Dziuba N. Hardy J. Lindahl P.A. Low-molecular-mass iron complexes in blood plasma of iron-deficient pigs do not originate directly from nutrient iron.Metallomics. 2019; 11: 1900-1911Crossref PubMed Google Scholar). The dominating features in the raw 6 K 0.05 T MB spectra (Fig. S5) were quadrupole doublets originating from contaminating deoxy and oxy FeII hemoglobin; these were simulated and removed. The resulting difference spectra were dominated by a magnetic feature which was simulated using the spin Hamiltonian parameters of di-ferric TFN (24Kretchmar S.A. Teixeira M. Huynh B.H. Raymond K.N. Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin.Biol. Met. 1988; 1: 26-32Crossref PubMed Scopus (24) Google Scholar). The only remaining spectral feature (Fig. 6, A and B respectively) was narrow absorption in the central region. Spectral noise was significant even after > 200 h of data collection. Spectral noise was too severe to firmly assign the absorption in the central region, but it could be approximately simulated by a quadrupole doublet with δ = 0.5 ± 0.1 mm/s and ΔEQ = 0.5 ± 0.1 mm/s. The parameters approximated those of FeIII aggregates such as the oxyhydroxide phosphate–associated nanoparticles found in diseased mitochondria (δ = 0.52 mm/s and ΔEQ = 0.63 mm/s; (25Miao R. Martinho M. Morales J.G. Kim H. Ellis E.A. Lill R. et al.EPR and Mössbauer spectroscopy of intact mitochondria isolated from Yah1p depleted Saccharomyces cerevisiae.Biochemistry. 2008; 47: 9888-9899Crossref PubMed Scopus (59) Google Scholar)). The intensity of this material was ∼ 2 × higher in the spectrum obtained from old versus young HFE mice. In animals with iron-overload diseases, NTBI concentration increases with age (26Lee S.M. Loguinov A. Fleming R.E. Vulpe C.D. Effects of strain and age on hepatic gene expression profiles in murine models of HFE-associated hereditary hemochromatosis.Genes Nutr. 2015; 10: 443Crossref PubMed Scopus (5) Google Scholar). Based on these observations, we hypothesized that NTBI was an FeIII aggregate in plasma. We considered that such aggregates formed due to the oxidizing, salty, pH-neutral conditions of plasma. To consider this further, we added acidic 57FeIII ions dropwise to blood plasma (to 250 μM) from 24 weeks HFE mice that had been fed a regular natural abundance-iron diet. The resulting spectrum (Fig. 6C) exhibited a broad quadrupole doublet again typical of 57FeIII aggregates (simulated in red). The minor magnetic species evident from the baseline was probably from added 57Fe that bound apo-TFN. We next prepared a “pseudo plasma FTS” solution and added acidic 57FeIII in similar fashion. The corresponding MB spectrum (Fig. 7D) was once again a quadrupole doublet with similar parameters (δ = 0.55 ± 0.02 mm/s and ΔEQ = 0.60 ±" @default.
• W4308295085 created "2022-11-10" @default.
• W4308295085 creator A5055094855 @default.
• W4308295085 creator A5069253317 @default.
• W4308295085 date "2022-12-01" @default.
• W4308295085 modified "2023-10-18" @default.
• W4308295085 title "Might nontransferrin-bound iron in blood plasma and sera be a nonproteinaceous high-molecular-mass FeIII aggregate?" @default.
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