Quantitation of ferritin iron in plasma, an explanation for non-transferrin iron.

In 33 patients with thalassemia and idiopathic hemochromatosis, plasma ferritin protein levels ranged from 36 to 5,850 micrograms/L. The iron content of this ferritin as determined by immunoprecipitation ranged from undetectable amounts to 507 micrograms/L. The mean iron content of ferritin protein in those and other subjects with plasma ferritin concentrations of over 1,000 was 6.8% +/- 2.7%. Plasma transferrin was usually saturated with iron in patients with measurable ferritin iron, but exceptions occurred. In studies using electrophoretic separation, it was shown that some ferritin iron moved to transferrin during in vitro incubation, whereas exchange in the opposite direction was extremely limited. Because some plasma ferritin iron was measured by the standard colorimetric plasma iron determination, these observations (a) indicate that plasma ferritin contains a significant amount of iron (b) indicate that a significant proportion of nontransferrin iron in individuals with nontransferrin iron as detected by standard plasma iron and total iron-binding capacity measurements is due to the presence of ferritin, and (c) suggest that large amounts of ferritin iron may affect the saturation of plasma transferrin.

Host-associated iron transfer factor in normal humans and patients with transfusion siderosis.

A low molecular weight iron-binding substance that promotes bacterial growth in vitro by increasing iron availability was identified in human blood and urine. Partial purification and physical characterization indicate that this factor is similar to the host-associated iron transfer factor (HAITF) previously isolated from mammalian tissue. HAITF was found to be significantly elevated in the blood of patients with thalassemia who have transfusional siderosis. The level of HAITF in the blood of these patients was also found to correlate with that of serum iron and serum glutamic-oxaloacetic transaminase (SGOT) but not with that of serum ferritin. Thus, elevated blood levels of HAITF may explain the increased susceptibility to infection seen in patients with iron overload. Its physiologic role, however, may involve the transport of iron within cells.

Preliminary toxicity findings in dogs and rodents given the iron chelator ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) (EDHPA).

Because of a projected pilot study with EDHPA in Cooley's anemia patients, animal studies with emphasis on reversibility of potential toxic signs were performed. Young dogs were treated iv with 6-18 mg/kg or orally with 30-240 mg/kg for 14 days followed by a 16-day recovery period. Drug-induced emesis, elevated BUN changes in kidney, spleen, and thymus weights diminished during recovery. One deceased dog exhibited nephrotoxicity consisting of tubular necrosis and deposition of the iron-EDHPA complex. The latter was observed in the excreta of survivors but kidney damage was not evident. Atrophy of the spleen and thymus in the deceased dog was consistent with less intense organ weight changes in recovered survivors. In the absence of morphologic changes after recovery, the precise effect on the immune system is unknown. The iv LD50 was 53 mg/kg for rats and mice. No rodent deaths occurred at an oral dose of 6000 mg/kg. An elevated BUN and changes in kidney, spleen, and thymus weights were confirmed in rodents given iv doses of 5-20 mg/kg or oral doses of 150-600 mg/kg for 5 days. It is cautioned that during the use of EDHPA derivatives that the functions of the renal and immune systems be monitored.

Acquisition of iron from transferrin regulates reticulocyte heme synthesis.

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.

Ferric pyridoxal isonicotinoyl hydrazone can provide iron for heme synthesis in reticulocytes.

We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxal isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20-200 microM 59Fe-PIH. The amount of 59Fe incorporated with 200 microM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 microM 59Fe concentration. After 60 min about 60% of the 59Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of 59Fe into heme by about 70%, indicating that 59Fe from 59Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate 59Fe from 59Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate 59Fe from 59Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.

Approaches to the standardization of serum unsaturated iron-binding capacity.

An important aspect in the development and use of serum unsaturated iron-binding capacity (UIBC) procedures is the standardization of reference serum. Spectrophotometric titration of serum transferrin is the oldest of the UIBC methods; however, the literature on this subject indicates that the technique is not being fully exploited and describes a variety of suboptimal conditions. A spectrophotometric titration protocol is described which gives excellent reproducibility with fresh, frozen, lyophilized, and stored serum samples. An indirect UIBC method utilizing differential chelation and ultrafiltration is also described. In this method Fe3+ -NTA (nitrilotriacetic acid), in excess of the anticipated UIBC, is added to a serum sample supplemented with NaHC03. The addition of EDTA (ethelenediaminetetraacetic acid) allows the mobilization of iron from nonspecific macromolecular sites. The ultrafiltrate is analyzed for iron, and the UIBC is calculated. The results were validated via an ultrafiltration titration which was compared with a spectrophotometric titration. The precision of this method is excellent.

[Total serum iron-binding capacity: problems posed by the use of commercial control sera]. / La capacité totale de fixation du fer par le sérum: problèmes posés par l'utilisation des sérums de contrôle du commerce

After recalling the conditions of fixation of iron on human native serum, the authors determined the TIBC on 15 commercial control sera after titration. The transferrin concentration was determined on 13 sera of human origin, by two immunochemical techniques. They were thus able to demonstrate certain sera including a chelator substance other than transferrin, their TIBC depends only on the experimental conditions; such sera are not usable for quality control. Lyophilisation seems to reduce the reactivity of sites of fixation. It is thus necessary to provide a large excess of iron and prolong the duration of contact.