Differential response of non-transferrin bound iron uptake in rat liver cells on long-term and short-term treatment with iron.

BACKGROUND: Uptake of non-transferrin-bound iron by the liver is important as a clearance mechanism in iron overload. In contrast to physiological uptake via receptor-mediated endocytosis of transferrin, no regulatory mechanisms for this process are known. This study compares the influence of long-term and short-term depletion and loading of hepatocytes with iron on the uptake of non-transferrin bound iron, its affinity, specificity and the interaction with the transferrin-mediated pathways. METHODS: Rats were fed iron-deficient, normal and 3,5,5-trimethylhexanoyl-ferrocene-containing diets to obtain livers with the corresponding desired status and the hepatocytes from these livers were used for transport studies. Hepatocytes from normal rats were depleted or loaded with iron by short-term treatment with desferrioxamine or ferric ammonium citrate, respectively. Uptake of non-transferrin bound iron was assayed from ferric citrate and from ferric diethylene triammine pentaacetate. RESULTS: Uptake of non-transferrin-bound iron in hepatocytes could be seen as consisting of a high-affinity (Km=600 nM) and a low-affinity component. Whereas in normal and in iron-starved rats the high-affinity component was more prominent, it disappeared altogether in hepatocytes from rats with iron overload resulting from prolonged feeding with TMH-ferrocene-enriched diet. Overloading also led to loss of inhibition by diferric transferrin, which occured in starved as well as normal cells. In contrast, short-term iron-depletion of isolated hepatocytes with desferrioxamine had only a weak stimulatory effect, whereas treatment with ferric ammonium citrate strongly increased the uptake rates. However, the inhibition by diferric transferrin also disappeared. In both cases, uptake of non-transferrin bound iron was inhibited by apotransferrin. CONCLUSIONS: Non-transferrin bound iron uptake in liver cells is apparently regulated by the iron status of the liver. The mode of response to iron loading depends on the method of loading in terms of time course and the form of iron used. It cannot be explained by the behavior of the iron regulatory protein, and it is complex, seeming to involve more than one transport system.

Late effects of bone marrow transplantation for thalassemia.

Long term effects of BMT in thalassemia were monitored in 33 patients transplanted between 1987 and 1995 and compared with 155 patients matched for age and treated during the same period with conventional therapy (CT). The incidence of fulminant sepsis and growth impairment was significantly higher in transplanted patients, whereas the occurrence of hypothyroidism, hypogonadism, and cardiopathy was higher in CT patients. For diabetes, liver disease, and severe infections, the differences were not statistically significant. After BMT we performed monthly erythrocytaferesis for iron removal in 23 (70%) patients, obtaining a complete normalization of iron stores in 91% of cases; among untreated patients, 60% had evidence of iron up to 8.3 years after BMT. Protection against poliovirus, tetanus, diphtheria, and hepatitis B has been lost in 74%, 47%, 78%, and 44%, respectively. After BMT a careful follow-up is needed to monitor and treat late transplant-related and thalassemia-related complications.

Nontransferrin-bound iron in serum of patients receiving bone marrow transplants.

Nontransferrin-bound iron (NTBI) and other parameters of iron status were measured in 40 patients undergoing bone marrow transplantation (BMT) prior to conditioning therapy (between day -10 and -7), at the time of BMT (day 0), and 2 weeks later (day + 14). Serum iron and transferrin saturation values were normal before conditioning therapy. At day 0 serum iron values were high and median transferrin saturation was 98% (changes in the values of both serum iron and transferrin saturation, p < .0001). Transferrin saturation values were still elevated 2 weeks posttransplant (day +14 vs. baseline values, p = .0001). Starting at low NTBI levels pretransplant (median 0.4 micromol/l, range 0-4.2 micromol/l, controls: < or = 0.4 micromol/l), all patients revealed high levels on day 0 (median 4.0 micromol/l, range 1.9-6.9 micromol/l, p < .0001) and 2 weeks posttransplant (median 2.7 micromol/l, range 0-6.2 micromol/l, p < .0001). These observations indicate that the plasma iron pool in patients undergoing BMT increases to a level at which the normal ability to sequestrate iron becomes exhausted and considerable amounts of NTBI appear in serum. This "free" form of iron can mediate the production of reactive oxygen species and may cause organ toxicity in the early posttransplantation period.

Ethane exhalation and vitamin E/ubiquinol status as markers of lipid peroxidation in ferrocene iron-loaded rats.

Organ damage caused by iron overload has been mostly attributed to iron-induced peroxidation of membrane lipids. Using the ferrocene iron-loaded rat model, we studied ethane exhalation as a direct marker of in vivo lipid peroxidation, as well as concentrations of alpha-tocopherol and ubiquinol 9/10 in liver and plasma as indirect markers of this process. The feeding of a diet enriched with 0.5% TMH-ferrocene up to 31 weeks resulted in a large increase in liver iron concentration to about 25 mg/g wet weight (w wt). At lower, predominantly hepatocellular liver siderosis, the breath ethane exhalation was dependent on dietary vitamin E (VitE) supplements (onset of ethane exhalation at liver-Fe > 2 mg/g w wt on vitE-restricted diet; > 5 mg Fe per gram on VitE-replete diet). At severe liver siderosis, breath ethane exhalation reached a maximum of approximately 8 nmol/kg/hr independent of VitE supplementation. Plasma as well as hepatic alpha-tocopherol decreased with progressive iron loading. In addition, a significant depletion in hepatic ubiquinol 9 and 10 was noted.

In vivo binding of radiocesium by two forms of Prussian blue and by ammonium iron hexacyanoferrate (II).

The effect of two forms of Prussian blue, soluble K3 Fe[Fe(CN)6] and insoluble Fe4[Fe(CN)6]3, and of ammonium iron hexacyanoferrate (II) (NH4Fe[Fe(CN)6] on intestinal radiocesium absorption was investigated in rats, pigs, and humans. In rats 5 mg of antidote administered 2 min before 134Cs tracer reduced radiocesium absorption to 2.4-6.3% of the oral dose. In pigs fed with Chernobyl-contaminated whey under normal feeding conditions for a 27 day period, radiocesium activity concentration was reduced from 360 Bq/kg in control animals to 10-30 Bq/kg by 5 g antidote/d. In man 1 g of oral Prussian blue diminished the cesium absorption from a 134Cs-labelled test meal to 5.6-6.4% of the controls. The inhibitory effects of colloidally soluble K3 Fe[Fe(CN)6] and of (NH4Fe[Fe(CN)6] were similar with slightly less inhibition by the insoluble Fe4[Fe(CN)6]3.

Bioavailability of iron and cyanide from 59Fe- and 14C-labelled hexacyanoferrates(II) in rats.

"Soluble" (KFe(III)[Fe(II)(CN)6]) and "insoluble Prussian blue" (Fe(III)4[Fe(II)(CN)6]3 labelled with 59Fe either in the ferric (Fe(III)) or ferro (Fe(II)) position and 14C in the cyanide group were synthesized and administered intraperitoneally or orally to adult female rats with normal body iron stores. Following i.p. injection of KFe[Fe(CN)6], the colloidal complex is disintegrated into ferric iron and hexacyanoferrate(II) anion almost completely. About 96% of the ferric iron was retained in the body. Nearly 90% of both ferrous iron and cyanide were excreted with the urine within 7 days after i.p. injection, indicating that most of the undissociated hexacyanoferrate(II) anion ([Fe(CN)6]4-) was excreted through the kidney. Only 9% of the ferrous iron from [Fe(CN)6]4- was found mainly in carcass, liver and gut. As the 59Fe/14C-ratios in organs were found close to 1.0, the dissociation of the hexacyanoferrate(II) anion can only be small in vivo. No detectable 14CO2-activity (less than 0.01%) was monitored in the breath of rats after i.p. injection of the 14C-labelled KFe[Fe(CN)6], also indicating that no significant amounts of cyanide were released after parenteral administration. After oral administration of the soluble and insoluble Prussian blue, 0.3-0.7% of the ferric iron was absorbed and retained mainly in carcass, liver and blood. Only 0.06-0.18% of the ferrous iron was absorbed and mostly excreted with the urine (0.05-0.15%), so that only 0.01-0.03% of the oral ferrous 59Fe was retained in the body after 7-10 days. Very small fractions of 14C-label from the 14CN-group of the soluble and insoluble hexacyanoferrate(II) were observed in the exhaled air (0.04-0.08% of the oral dose). From the 14CO2-exhalation, the 14C-urine excretion and the distribution of iron in blood and organs it can be concluded that the hexacyanoferrate(II) moiety disintegrated only to a small extent in the intestinal tract after oral administration. From a dose of 36 mg hexacyanoferrate(II)/kg, an amount of free (non-complex bound) cyanide can be calculated which is in maximum two orders of magnitude below the LD100-level. Thus, the very low bioavailability of iron and cyanide from hexacyanoferrate(II) compounds after oral application is demonstrated in rats. In the case of a severe nuclear accident, appropriate doses of "soluble" and "insoluble" Prussian blue can be used as safe and effective antidote against radiocaesium contamination.

Bioavailability of iron and cyanide from oral potassium ferric hexacyanoferrate(II) in humans.

After oral administration of 500 mg KFe[Fe(CN)6] labelled with 59Fe either in the ferric or ferrous position and with 14C in the cyanide group only 0.22% of the FeII and less than 0.04% of the FeIII were absorbed in three male volunteers. Only 2 mg non-complex bound 14C-labelled cyanide (0.03 mg CN-/kg body wt) were absorbed from 500 mg [14C]KFeHCF, which is about a factor of 20-100 below the lethal dose in humans (0.5-3.5 mg CN-/kg body wt). Therefore, iron(III) hexacyanoferrates(II) can be considered as safe antidotes, i.e. for inhibiting the intestinal absorption of radiocaesium or for accelerating the excretion of already absorbed 134/137Cs in the case of a severe nuclear accident.

Intestinal absorption of iron from 59Fe-labelled hexacyanoferrates(II) in piglets.

The intestinal absorption of 59Fe and 14C from hexacyanoferrates(II) was studied in piglets. KFeIII[FeII (CN)6] (I) and FeIII4[FeII(CN)6]3 (II) were labelled with 59Fe both in the Fe(III)-position (outside the complex anion, a) or in the Fe(II)-position (hexacyanoferrate anion, b). Labelling of the Fe(III)-position resulted in a 59Fe-absorption of 1.47% (Ia) and 1.34% (IIa), as judged by the 59Fe whole-body-retention measurement 14 days after oral administration. Even smaller amounts, 0.20% from Ib or 0.15% from IIb of the 59Fe-dose were absorbed and retained from the hexacyanoferrates(II) labelled in the Fe(II)-position. No 14CO2 was detected in the expired air of piglets after oral application of Fe4[59Fe(14CN)6]3, indicating that the amount of incorporated free cyanide ions can only be extremely small or even nil.