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.

Active uptake of serotonin as viability parameter of stored platelets.

The platelet-plasma serotonin (5-HT) exchange in different apheresis products and platelet concentrates (PCs) from buffy coats was studied for quality control over a period of 7 days. In PCs with steadily decreasing pH due to inappropriate gas exchange the platelet 5-HT concentration increases significantly (> 10% positive netto uptake) during the 1st day of storage. Thereafter platelet serotonin drops rapidly with t1/2 = 1 day at pH < 6.5, but platelet counts were constant and 5-HT uptake was inhibited concomitantly. Plasma 5-HT increases as pH decreases. This can be enforced by competitive inhibition of serotonin uptake due to imipramine which also moderates the total 5-HT loss. However, in PC with stable pH the operative 5-HT pump keeps steady-state conditions. The efficacy of the transmembrane uptake mechanism can be adequately monitored by the assessment of the 5-HT platelet/plasma ratio as a new platelet viability parameter.

205Bi/206Bi cyclotron production from Pb-isotopes for absorption studies in humans.

Pb(p, xn) thick target excitation functions were measured in the energy range 10-38 MeV in order to optimize the production of isotopically pure radiobismuth from natPb, 206Pb, and 207Pb. Additionally, the decay of Po-isotopes from deuteron irradiation of natural bismuth (209Bi) was exploited for radiobismuth production. 205Bi was produced from 206Pb at 20 MeV with only 2% of 206Bi at 4 weeks post irradiation. Bismuth compounds as used in the treatment of peptic ulcer were labeled with 205Bi for absorption studies in animals and subjects.

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.

Bismuth absorption from 205Bi-labelled pharmaceutical bismuth compounds used in the treatment of peptic ulcer disease.

The absorption of bismuth from five 205Bi-labelled pharmaceutically used bismuth compounds was studied in man. From single oral doses of all compounds under investigation only less than 0.1% bismuth was absorbed and excreted with the urine. A significantly higher absorption was observed from the colloidal bismuth subcitrate (0.042% of the dose) and the basic bismuth gallate (0.038%) than from the basic bismuth salicylate, nitrate, and aluminate (0.005-0.002%). No retention of bismuth in the whole body was found from the single dose experiment. The biologic fast-term half-lives of absorbed bismuth were calculated to be 0.12 and 1.5 days.

Inhibition of intestinal absorption and decorporation of radiocaesium in humans by hexacyanoferrates(II).

The effect of hexacyanoferrate(II) preparations, KFe[Fe(CN)6], (KFeHCF) anol Fe4[Fe(CN)6]3, (FeHCF) on intestinal radiocaesium absorption was studied in two male volunteers. The 134Cs absorption was decreased from 100 to 3-10% when 500-1000 mg KFeHCF or FeHCF were administered 10 min before the 134Cs-labelled test meal. However, when HCF was administered simultaneously with the test meal, the 134Cs absorption was decreased to only 38-63%. The biological half-time of previously absorbed 134Cs was reduced from 106 (73) to 44 (46) days by daily administration of 3 times 0.5 g KFeHCF. The 134Cs dose conversion factors lie below the values recommended by IRCP 30, indicating that the IRCP model represents a cautious description of the Cs biokinetics in our study.

Bioavailability of bismuth from 205Bi-labelled pharmaceutical oral Bi-preparations in rats.

The bioavailability of 205Bi from various 205Bi-labelled pharmaceutical oral bismuth preparations was studied in rats. The intestinal absorption, calculated from 205Bi whole body retention and accumulated 205Bi urinary excretion, was small in general, but significantly higher (0.26-0.33% of dose) from oral bismuth citrates (basic bismuth citrate, colloidal bismuth subcitrate) as compared to basic bismuth nitrate, salicylate, gallate, and bismuth aluminate (0.04-0.11% of dose). After oral administration, the retained bismuth was mainly accumulated in the kidney, followed by bone, red blood cells and the lung. The whole body retention, faecal and urinary excretions of 205Bi were described by a three-compartment model. Biological 205Bi half-lives of 10, 36 and 295 h were derived in rats.

Efficacy of different hexacyanoferrates(II) in inhibiting the intestinal absorption of radiocaesium in rats.

The inhibitory effect of various oral doses of different hexacyanoferrate(II) compounds (HCF) and the influence of the time interval of HCF-administration on intestinal 134Cs-absorption was studied in rats. Optimum inhibition was obtained by administration of HCF together with or 2 min before oral 134Cs loading. Using appropriate low amounts (0.1-0.5 mg) of the different HCF compounds, the inhibitory effect increased in the sequence KZnHCF less than KCuHCF less than FeHCF less than KCoHCF = KNiHCF less than NH4FeHCF = KFeHCF. Oral administration of 5 mg (0.5 mg) of KFeHCF, together with 134CsCl loading, reduces 134Cs-absorption from 41% (control) to 0.8% (2.8%). Zinc-, copper-, cobalt, and nickel hexacyanoferrates(II), despite showing a high caesium sorption capacity in vitro, were less effective in rats and are not suited for in vivo application, also because they may produce toxic side effects. As a consequence, the orally administered colloidal-soluble iron(III) hexacyanoferrates(II) (NH4Fe[Fe(CN)6] and KFe[Fe(CN)6]) have to be considered as the most valuable countermeasure against radiocaesium absorption for humans and domestic animals in the case of a severe nuclear accident in the future. Manganese oxide, a non-hexacyanoferrate(II) compound with known in vitro caesium binding capacity, showed no inhibitory effect on radiocaesium absorption in rats.

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.

In-vitro culture of human chondrocytes from adult subjects.

An agarose gel matrix was utilized to grow chondrocytes from human donors of various ages in cell culture. The chondrocytes produced the pericellular matrix characteristic for such cells and synthesized collagen type II as well as glyco-saminoglycans. The latter exhibit the typical distribution pattern of the respective articular cartilage matrix. The electron-microscopic appearance of the cultured chondrocytes closely resembles that of chondrocytes in sections of the original cartilage.

[Tissue proliferation by thrombocyte growth factor]. / Gewebsproliferation durch Thrombozytenwachstumsfaktor.

An essay for determining the growth stimulating activity of human platelets has been established. With the aid of this system a mitogenic factor from crude platelet extract was isolated. Besides a stimulation of DNA-synthesis an enhanced synthesis of collagen and glycosaminoglycans is induced by the platelet growth factor.

The isolation and activity of growth-stimulating factors from human platelets.

Optimal conditions for determining the mitogenic activity of platelet growth factor in a homologous system (human palmar fascia fibroblasts/human platelet growth factor) were established. With the aid of this system it was possible to test the active fraction from crude platelet extract following affinity chromatography on heparin-Sepharose, chromatofocusing and molecular sieve filtration. This fraction has an isoelectric point of pH greater than 9 with a molecular mass of about 12000 daltons and reacts with beta-thromboglobulin antiserum. The addition of protease inhibitors proved essential for the isolation of the active growth factor, in order to prevent proteolytic degradation to a biologically inactive substance that is immunologically identical to beta-thromboglobulin. In addition to stimulation of DNA synthesis, the isolated growth factor induces enhanced synthesis of collagen and glycosaminoglycans in fibroblasts in vitro.

Extracellular metal-free corrinoids from Rhodopseudomonas spheroides.

Rhodopseudomonas spheroides when grown on a medium deficient of cobalt excretes significant amounts of descobaltocorrinoids into the culture broth. If grown in the presence of 4 microM CoCl2 only intracellular cobalt-containing corrinoids are detected. The extracellular corrinoids have been identified as hydrogenobyrinic acid c-amide and hydrogenobyrinic acid a,c-diamide which are accompanied by varying amounts of the corresponding 13-epicorrinoids. The latter, which could not be separated in the metal-free form, are artifacts arising from the excreted descobaltocorrinoids under the alkaline conditions of the culture broth. The red metal-free corrinoids have been converted into the corresponding cobalt-containing corrinoids and were characterized by their CD, UV, 1H NMR, and 13C NMR spectra. Insertion of cobalt into the fraction of hydrogenobyrinic acid c-amide has yielded small amounts of a cobalt-containing corrinoid, for which the structure of 18,19-didehydrocobyrinic acid c-amide is suggested. A 14C-labeled specimen of hydrogenobyrinic acid a,c-diamide was obtained by feeding [14C]acetate to growing cultures of R. spheroides. Experiments with broken cell systems of Propionibacterium shermanii and R. spheroides have indicated that metal-free corrinoids are not precursors of the cobamides. The results are discussed with respect to the final steps of the biosynthesis of vitamin B12.