Effect of alpha-lipoic acid preparations on bilirubin and transferrin levels and ferritin iron and transferrin iron content

The influence of ethylendiamine salt of alpha-lipoic acid on the indices of iron metabolism in patients with occupational pathologies has been studied using quantitative electron spin resonance spectroscopy. In the cases of treatment in the patients suffering from hyperferremia the decrease in transferrin iron concentration in the whole blood and plasma occurs correlating with the enhancement of iron excretion from urine and decline of bilirubin level in serum. We have found that the preparation chelates iron from iron (III)-citrate complex and form stable iron (III) complexes. The conclusion is that the positive effects of lipoic acid preparation in the patients with hyperferremia at least partially could be associated with normalization of iron exchange and reduction in the labile iron pool.     

Uptake of iron from transferrin by isolated hepatocytes

Isolated rat hepatocytes containing 0.56-1.79 micrograms iron/10(6) cells and with an intracellular ATP concentration of 3-4 mM, accumulate iron from transferrin linearly with time for at least 3 h. At 37 degrees C the rate of uptake amounts to 0.3-0.7 pmol/mg cell protein per min. The uptake reaches a saturation level of 21-40 pmol/mg cell protein per h at 2.2 microM iron. At 5 degrees C the uptake does not increase over the time of incubation. Uptake of iron, but not binding of transferrin is increased 4-5-fold at oxygen concentrations 10-20 microM. At oxygen concentrations beyond these limits iron uptake is decreased. Iron taken up at low oxygen concentrations can be chelated by bathophenanthroline and bathophenanthroline disulphonate , but only if the chelators are present during the uptake experiments. The results suggest that iron uptake from transferrin by hepatocytes in suspension involves reductive removal of iron.     

Effect of iron transferrin on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

In serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, iron transferrin causes enhanced uptake of both tritiated thymidine and tritiated uridine over that seen with only phytohemagglutinin. This effect is specific for the iron transferrin complex, no enhancement produced by either free iron(III) or apotransferrin. Iron bound to transferrin is quantitatively taken up by stimulated lymphocyte cultures, while under similar conditions only 10% of transferrin-bound zinc is incorporated. The relative specificity of action of iron and zinc on nucleic acid synthesis is discussed.     

The effect of transferrin saturation on internal iron exchange

Radioiron was introduced into the intestinal lumen to evaluate absorption, injected as nonviable red cells to evaluate reticuloendothelial (RE) processing of iron, and injected as hemoglobin to evaluate hepatocyte iron processing. Redistribution of iron through the plasma was evaluated in control animals and animals whose transferrin was saturated by iron infusion. Radioiron introduced into the lumen of the gut as ferrous sulfate and as transferrin-bound iron was absorbed about half as well in iron-infused animals, and absorbed iron was localized in the liver. The similar absorption of transferrin-bound iron suggested that absorption of ferrous iron occurred via the mucosal cell and did not enter by diffusion. The decrease in absorption was associated with an increase in mucosal iron and ferritin content produced by the iron infusion. An inverse relationship (r = -0.895) was shown between mucosal ferritin iron and absorption. When iron was injected as nonviable red cells, it was deposited predominantly in reticuloendothelial cells of the spleen. Return of this radioiron to the plasma was only 6% of that in control animals. While there was some movement of iron from spleen to liver, this could be accounted for by intravascular hemolysis. Injected hemoglobin tagged with radioiron was for the most part taken up and held by the liver. Some 13% initially localized in the marrow in iron-infused animals was shown to be storage iron unavailable for hemoglobin synthesis. These studies demonstrate the hepatic trapping of absorbed iron and the inability of either RE cell or hepatocyte to release iron in the transferrin-saturated animal.     

The effect of acid pH and citrate on the release and exchange of iron on rat transferrin

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1 · 10^?3 M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

The effect of acid pH and citrate on the release and exchange of iron on rat transferrin.

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1.10(-3) M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

Coordination chemistry of microbial iron transport compounds: rhodotorulic acid and iron uptake in Rhodotorula pilimanae.

The mechanism by which iron uptake is facilitated by the siderophore rhodotorulic acid (RA) in the yeast Rhodotorula pilimanae was investigated with radioactively labeled Fe and RA and kinetically inert, chromic-substituted RA complexes. The weight of the evidence supports a model in which RA mediates iron transport to the cell but does not actually transport iron into the cell. It is proposed that RA exchanges the ferric ion at the cell surface with a membrane-bound chelating agent that completes the active transport of iron into the cell. Uptake of 55Fe in ferric rhodotorulate was much more rapid than uptake of RA itself. Two exchange-inert chromic complexes of RA showed no uptake.     

Intracellular processing of transferrin and iron by isolated rat hepatocytes.

Transferrin bound by isolated rat hepatocytes is rapidly endocytosed and enters a compartment of low density. Little was found associated with the lysosomes, even though the protein was subsequently lost from the cells. Iron entering the cells on transferrin was subsequently found in a number of intracellular components: transferrin, haem, ferritin and a residual fraction. After 2 h incubation with 59Fe-transferrin almost 70% of the iron was in ferritin, and this proportion increased to 80% during a 'chase' experiment. Residual iron, because of its rapid increase at the start of the incubation and its decline during the 'chase', probably represents an intracellular transit pool, which at steady state was present at 23 pg/10(6) cells.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Hydroxamate siderophore mediated iron uptake in E. coli: stereospecific recognition of ferric rhodotorulic acid

Ferric ion uptake via the fhuE receptor of E. coli is stereospecific; it prefers the delta-absolute configuration of the metal complexes. The importance of the metal center and its adjacent functionalities for the stereospecific recognition of ferrichromes via TonA is discussed.     

Effect of transferrin on enhancing bioavailability of iron

Transferrin was isolated and purified from bovine plasma. An intestinal segment in situ experiment showed that 19.2% of injected iron was absorbed when FeCl(3) (80 microg Fe/ml) was injected into a duodenum segment of iron-deficient rats. With addition of 10 and 20 mg of purified transferrin/ml, however, ratios of absorbed iron through duodenum segments were significantly increased to 52.7 and 57.9%, respectively. After transferrin-rich extract was isolated by batch type ion exchange chromatography, a soluble ferric complex of the transferrin extract was prepared by adding ferric salts to transferrin extract followed by dialysis, sterilization, and freeze drying. Results of the animal experiment for comparing bioavailabilities of different irons showed that irons in Fe-transferrin extract was most efficiently absorbed and incorporated into hemoglobin generation in anemic rats.     

Protection of human and murine hepatocytes against Fas-induced death by transferrin and iron

Recent studies in a murine model show that transferrin (Tf) interferes with Fas-mediated hepatocyte death and liver failure by decreasing pro-apoptotic and increasing anti-apoptotic signals. We show here in vitro in murine and human hepatocyte cell lines and in vivo in mice that Fas-induced apoptosis is modulated by exogenous Tf and iron. The results obtained with iron-free Tf (ApoTf), iron-saturated Tf (FeTf), and the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in its iron-free and iron-saturated (FeSIH) forms indicate that apoptosis-modulating effects of Tf are not mediated by iron alone. Both the Tf molecule and iron affect multiple aspects of cell death, and the route of iron delivery to the cell may be critical for the final outcome of cellular Fas signaling. Survival of hepatocytes ‘stressed’ by Fas signals can be manipulated by Tf and iron and may be a target for prophylactic and therapeutic interventions.     

The mechanism and specificity of iron transport in Rhodotorula pilimanae probed by synthetic analogs of rhodotorulic acid

The yeast Rhodotorula pilimanae produces the dihydroxamate siderophore rhodotorulic acid (RA) in prodigious amounts when starved for iron. Synthetic dihydroxamate analogs of RA have been prepared in which the diketopiperazine ring of RA is replaced by a simple chain of n methylene groups. It is found that R. pilimanae is able to accumulate iron using these achiral complexes, as well as from simple monohydroxamate analogs, at rates comparable to those of RA. While the Fe2RA3 complex does not enter the cell, there is a receptor system whose geometric requirements for siderophore recognition have been probed using analogs. In contrast to mono- or dihydroxamate ligands, the trihydroxamate siderophores such as ferrioxamine B are completely ineffective at delivering iron to R. pilimanae. This is ascribed to the greater stability of these complexes, which blocks release of the Fe(III) in a ligand exchange process that is required for uptake. To explore whether this ligand exchange involves redox catalysis, Ga(III) was substituted for Fe(III). The gallium was taken up at rates near those of iron and were also energy-dependent, as determined by metabolic inhibition with KCN.     

Identification of an iron uptake system specific for coprogen and rhodotorulic acid in Escherichia coli K12

With the lac operon fusion technique, mutants were isolated in two genes that specify two outer membrane proteins designated FhuE (76 K) and Fiu (83 K). The synthesis of both proteins was increased under low iron growth conditions. The FhuE-protein was shown to be necessary for iron uptake via coprogen, an iron chelator produced by certain fungi, e.g. Neurospora crassa. In addition to fhueE the genes fhuCDB, tonB and exbB were necessary for iron coprogen uptake. The gene fhuE was mapped between kdp and gltA near 16 min on the genetic map of E. coli K12, while gene fiu was mapped near 18 min between chlA and chlE. Nor iron transport system could be assigned as yet to the Fiu protein.     

Effects of iron and desferrioxamine on Rhizopus infection

To investigate the association among iron, desferrioxamine, and a Rhizopus infection, the influence of iron and/or desferrioxamine on experimental mucormycosis in mice was examined. All mice pretreated with iron, desferrioxamine, or a combination of iron and desferrioxamine died within 5 days after the inoculation of R. oryzae. In the mice fungal lesions were observed in the brain which resembled human cerebral mucormycosis. By contrast, the mortality in the control mice with R. oryzae was 20% through the 3-week experimental period. Therefore, it was demonstrated that iron as well as desferrioxamine administration markedly promotes the growth of R. oryzae. The increased susceptibility to R. oryzae was considered to be due to increased serum iron in the animals pretreated with iron only; however, pretreatment with desferrioxamine did not affect the amount of serum ion. Thus, the data suggest that desferrioxamine acts as a siderophore to R. oryzae and exerts an adverse effect on mucormycosis. This study has shown that the presence of iron and desferrioxamine enhances the virulence and pathogenicity of R. oryzae by serving as a growth factor.     

A mathematical model of iron chelation with desferrioxamine

Removal of body iron is a severe clinical problem in patients affected by hemochromatosis or iron-loading anemias. Desferrioxamine (DFO) is the most potent artificial iron-chelating agent. This work deals with the problem of describing DFO action in man by means of a mathematical model, formulated on the basis of the current knowledge about DFO and iron metabolism. Since most clinical data are concerned with DFO-induced urinary iron excretion, only this process, which involves iron stored in reticuloendothelial (RE) cells, was described in detail. Most of the parameters of the model were estimated from data obtained by studying the single processes involved in iron chelation with DFO. A computer simulation study then allowed us to assign meaningful values to the remaining parameters. Different DFO treatments were simulated, and the results obtained seem to show us that the model can reproduce the main experimental findings and the relationship between the amount of chelated iron and the iron status of the patient.     

Primary culture of chicken hepatocytes in serum-free medium (pH 7.8) secreted albumin and transferrin for a long period in free gas exchange with the atmosphere

To study the liver functions of chicken, we examined the primary culture of chicken hepatocytes, and found an easy method of long-term culture with free atmosphere exchange. Chicken hepatocytes were obtained by collagenase perfusion and cultured at 37°C as a monolayer without substratum in serum-free L-15 medium (pH 7.8) with free atmosphere exchange. The amounts of albumin and transferrin in medium were assayed by ELISA. The culture of chicken hepatocytes was maintained in the serum-free L-15 medium (pH 7.8) at 37°C with free atmosphere exchange for 20 days. The amount of albumin secreted in the medium decreased to low levels early in culture; however, this was followed by marked increase from day 9 to day 17 of culture. The amount of transferrin was constant until day 6, then it too increased considerably with further culture. We reported an easy method for the simple monolayer culture of chicken hepatocytes in serum-free L-15 medium (pH 7.8) with free atmosphere exchange over an extended period. Expression of liver-specific functions, viz. albumin and transferrin synthesis, was observed after 1 week of culture.     

Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.