Inge Romslo

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.

Rescue after Intermediate and High-Dose Methotrexate: Background, Rationale, and Current Practice

Pharmacologic rescue methods used in combination with intermediate and high-dose methotrexate therapy are reviewed, with special emphasis on rescue with nucleosides and folinic acid. The mechanism of action, pharmacokinetics, and clinical applications of the rescue agents are described in detail in view of the literature and also of the own findings of the authors. In spite of the promising results of the in vitro studies and in vivo experiments in animal models, the clinical value of thymidine as a rescue agent remains to be determined. Currently, the only indication to use thymidine instead of folinic acid following high-dose methotrexate is to prevent toxicity related to extremely high methotrexate levels in patients with delayed elimination of methotrexate. In spite of the widespread application of folinic acid rescue, the exact mechanism of its action is not fully understood. The rescue dose and schedule in the majority of clinical protocols is empirical, and the start of the rescue administration is too early, allowing less than 36 to 42 hours of exposure to methotrexate. Clinical and laboratory findings indicate that while the early start of FA administration is unnecessary for protecting normal cells, it is potentially dangerous in terms of reduction of the antitumor effect of methotrexate. Our findings suggest that less than the most widely used 12-15 mg/m2 per dose rescue may be sufficient in preventing methotrexate related toxicity in patients with normal elimination of the drug. In addition, reducing the dose of the rescue may be beneficial to achieve better therapeutic results with high-dose methotrexate. Due to methodological problems, the pharmacokinetics of folinic acid rescue has not been excessively studied in humans. Recent data indicate that the pharmacokinetics of folinic acid in children is characterized by great intra- and interpatient variability. The effect of food on the bioavailability of folinic acid has not yet been studied, though it is most frequently administered orally. The introduction of the pure l-stereoisomer of the rescue agent in the clinical practice may eliminate potential interactions with the d-isomer, and may also simplify the introduction of therapeutic drug monitoring for folinic acid as well. This could lead to more rational clinical use of folinic acid as a rescue agent following intermediate and high-dose methotrexate therapy.

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.

Day-to-day variations in serum iron, serum iron binding capacity, serum ferritin and erythrocyte protoporphyrin concentrations in anaemic subjects.

Day‐to‐day variations in serum iron, serum iron binding capacity, serum ferritin and erythrocyte protoporphyrin were determined on 2 successive days in 48 patients with anaemia. The correlation coefficients between the paired determinations were 0.86, 0.89, 0.95 and 0.95, and the day‐to‐day coefficients of variation (in per cent) were 33, 11, 12 and 13 for serum iron, serum iron binding capacity, erythrocyte protoporphyrin and serum ferritin, respectively. Thus, in patients with anaemia, day‐to‐day variations in serum iron, serum iron binding capacity, erythrocyte protoporphyrin and serum ferritin are at least as high as in healthy controls. The results indicate important limitations in the use, particularly, of serum iron in the clinical investigation of anaemia.

Pyrophosphate as a ligand for delivery of iron to isolated rat-liver mitochondria

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The capacity of the mitochondria to accumulate iron is higher than the capacity of pyrophosphate to mobilize iron from transferrin: with ferric-iron-pyrophosphate as iron donor, iron uptake and heme synthesis are about 10-times that at corresponding concentrations of iron-transferrin plus pyrophosphate. Uptake of iron from ferric-iron-pyrophosphate depends on a functionary respiratory chain and involves reductive cleavage of the ferric-iron-pyrophosphate complex. Apotransferrin inhibits uptake of iron from ferric-iron-pyrophosphate by competing with the mitochondria for iron. The results focus on pyrophosphate as a possible candidate for intracellular iron transport.

Variations in serum erythropoietin and transferrin receptor during phlebotomy therapy of hereditary hemochromatosis: A case report

Abstract:  Serum levels of transferrin receptor and erythropoietin were determined in 2 patients with hereditary hemochromatosis undergoing phlebotomy therapy. The objective of the study was to determine changes in serum transferrin receptor and serum erythropoietin occurring during therapy, and to investigate if such changes could be useful to monitor the therapy. The study showed that serum transferrin receptor, and to a lesser extent serum erythropoietin, may be better parameters than serum ferritin as indicators of when phlebotomy should be discontinued. The most sensitive parameter, however, appeared to be the serum transferrin ratio.

Iron uptake and heme synthesis by isolated rat liver mitochondria. Differic transferrin as iron donor and the effect of pyrophosphate

Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ.

The effect of brief illumination on intracellular free calcium concentration in cells with 5-aminolevulinic acid-induced protoporphyrin IX synthesis

The effect of illumination on intracellular free calcium concentration, [Ca2+]i, was studied in a cell line (WiDr cells) derived from a primary adenocarcinoma of the rectosigmoid colon. In these cells the biosynthesis of protoporphyrin IX was stimulated by 5-aminolevulinic acid to reach levels of 600-700 pmol of protoporphyrin IX per mg cell protein. A brief (1-min) exposure of the cells to light (70% of light energy at 340-380 nm) resulted in an increase in [Ca2+]i. This increase was not reversible over a period of at least 20 min following illumination. Elevation of [Ca2+]i most probably represented an influx of calcium ions from the medium to the cell, since it was completely abolished in the presence of extracellular EGTA. The increased [Ca2+]i did not reflect general membrane damage, as determined by trypan blue staining as well as measurement of the intercalation of ethidium bromide into cellular DNA, and neither did the sustained elevation of [Ca2+]i lead to any substantial loss of clonogenicity following illumination of protoporphyrin-containing cells. Together these results indicate that an increased [Ca2+]i level is not per se a cause of cell death during photodynamic therapy.

Determination of porphyrins by high performance liquid chromatography: fluorescence detection compared to absorbance detection

A comparison is made between determination of porphyrin methyl esters by high performance liquid chromatography (HPLC) with absorbance detection and with fluorimetric detection. Detection limits with absorbance detection vary from 0.8 pmol for 2-COOH-porphyrins to 5 pmol for 8-COOH-porphyrins. With fluorometric detection the corresponding figures are 0.04 pmol and 0.4 pmol. Fluorimetric detection also has a better reproducibility, and it is more specific than absorbance detection. The use of HPLC with fluorimetric detection thus permits rapid, highly efficient and specific quantitative detection and identification of porphyrins in complex biological samples.

Uptake of iron from transferrin by isolated hepatocytes. Relationship to cellular energy metabolism

The mechanism by which utilization of transferrin-bound iron is linked with cellular metabolism has been studied in isolated rat hepatocytes. The initial binding of transferrin to the hepatocyte is not dependent on metabolic energy, but the subsequent progressive binding of transferrin and uptake of iron depend on metabolic energy and the drainage of reducing equivalents from the respiratory chain. When respiration is completely blocked with cyanide a limiting energy level for the uptake of iron is found at an intracellular concentration of ATP of approximately 0.2 mmol/l. The iron uptake process utilizes ATP hydrolysis, substrate oxidation and dissipation of ionic gradients as energy sources interchangeably.