Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells.

OBJECTIVES: To generate non-haematopoietic tissues from mobilized haematopoietic CD133(+) stem cells. MATERIALS AND METHODS: Mobilized peripheral blood CD133(+) cells from adult healthy donors were used. In vitro ability of highly enriched CD133(+) cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. RESULTS: We found that a recently identified population of CD45(+) adherent cells generated in vitro after culture of highly purified CD133(+) cells for 3-5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. CONCLUSION: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.

Niemann-Pick disease type A and B are clinically but also enzymatically heterogeneous: pitfall in the laboratory diagnosis of sphingomyelinase deficiency associated with the mutation Q292 K.

This study describes a diagnostic pitfall in the laboratory diagnosis of patients with sphingomyelinase deficiency (SMD; Niemann-Pick disease types A and B; NPA and NPB), in cases where sphingomyelinase activity was not determined with sphingomyelin as the natural enzymic substrate. Four of 24 SMD patients studied had falsely normal or enhanced activity, when a so-called artificial sphingomyelinase substrate, 2-N-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine (HNP), was used, whereas SMD was clear with the sphingomyelin substrate. Those four patients had the Q292 K mutation of the acid sphingomyelinase gene (SMPD1) on at least one allele. Three of the four patients (no data available from one) experienced only late-infantile or juvenile, though distinct, neurological involvement, where learning disabilities, hypo- or areflexia or mild ataxia were initial signs. The laboratory pitfall with HNP substrate, which is used in many laboratories, raises the risk that some SMD patients are overlooked, and it prevents the consideration of a late-manifesting neurological course in some patients as well as the planning of enzyme substitution therapy in non-neurological SMD (NPB) patients. Since classical NPB is very rare, it is suggested that SMD patients with late- or mild-manifesting neurological symptoms should better be assigned to additional SMD subgroups than grouped with NPB.

Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase-containing liposomes.

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)-containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections.

Analysis of metabolites of glucose pathways in human erythrocytes by analytical isotachophoresis.

The aim of this study was to establish an isotachophoretic (ITP) method for the determination of the main compounds of glycolysis in human erythrocytes in order to analyze the influence of different glucose concentrations (mimicking the situation in diabetes mellitus) on this pathway. Samples for ITP were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration (Mr cut-off: 5000). All the main compounds of glycolysis were characterized by ITP. The influence of different glucose concentrations on the main compounds of the energy metabolism (ATP, ADP, lactate, pyruvate) and 2,3-diphosphoglycerate were analyzed in short- and long-time incubations.

6-fluorodopamine selectively destroys neuroblastoma cells expressing the noradrenaline transporter.

BACKGROUND: 6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells. PROCEDURE: We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells. RESULTS: Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA. CONCLUSIONS: We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells.

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid.

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.

Neuroblastoma cells expressing the noradrenaline transporter are destroyed more selectively by 6-fluorodopamine than by 6-hydroxydopamine.

6-Hydroxydopamine (6-OHDA) has been used for lesioning catecholaminergic neurons and attempted purging of neuroblastoma cells from hematopoietic stem cells in autologous bone marrow transplantation (ABMT). Neurotoxicity is mediated primarily by reactive oxygen species. In ABMT, 6-OHDA, as a purging agent, has been unsuccessful. At physiological pH it autooxidizes before targeted uptake, resulting in nonspecific cytotoxicity of nontarget cells. A catecholamine analogue, similar to 6-OHDA but with a lower rate of autooxidation enabling uptake by target cells, is thus required. Electron paramagnetic resonance spectra in this study show that 6-fluorodopamine (6-FDA) hydrolyzes slowly to 6-OHDA at physiological pH. Oxygen consumption, H(2)O(2), and quinone production are found to be intermediate between those of 6-OHDA and dopamine (DA). Relative neurotoxicity of these compounds was assessed by cell viability and DNA damage in the human neuroblastoma lines SH-SY5Y and SK-N-LO, which express and lack the noradrenaline transporter, respectively. Specific uptake of DA and 6-FDA by SH-SY5Y cells was demonstrated by competitive m-[(131)I]iodobenzylguanidine uptake inhibition. The competition by 6-OHDA was low owing to rapid autooxidation during incubation with equal toxicity toward both cell types. 6-FDA toxicity was preferential for SH-SY5Y cells and reduced in the presence of desipramine, a catecholamine uptake inhibitor. We demonstrate that 6-FDA cytotoxicity is more specific for cells expressing catecholamine reuptake systems than is 6-OHDA cytotoxicity.

Detection by ELISA of low transferrin levels in bronchoalveolar secretions of preterm infants.

Transferrin levels in bronchoalveolar secretions (BAS) are very low compared to serum levels in humans. For the exact measurement of transferrin concentrations in BAS a very sensitive assay was developed as a double sandwich enzyme immunoassay using the combination of a polyclonal and a monoclonal antibody against human transferrin. The measurable range of the assay was 1.5 to 100 ng/ml of human transferrin. The lowest measurable value was 0.84 ng/ml and the sensitivity of the assay was 0.88 ng/ml. The coefficient of variation was 14.1% for 25 ng/ml (intra-assay) and 11-20% (inter-assay). The levels measured in 123 samples of BAS of preterm infants ranged between 0.03 and 8.93 (microgram/microgram secretory component (SC)). The determination of transferrin in BAS of preterm infants is helpful in determining oxidative damage, e.g. the availability of free iron, in the neonatal lung. The transferrin concentration in BAS of neonates who recovered from respiratory distress syndrome (RDS) in the first six days of life was 0.48 compared to 0.52 ((microgram/microgram SC), median range) for infants who developed chronic lung disease.

Effect of amifostine on neuroblastoma during high dose chemotherapy: in vivo and in vitro investigations.

Amifostine (Ethyol, WR-2721) has been clinically used in combination with high dose therapy of neuroblastoma stage 4 with melphalan, carboplatin and VP-16 in 14 patients. The amifostine group was compared to a historical control group of 24 comparably-treated patients. There were no significant differences regarding the time of hematological recovery, the duration of hospitalization, the duration of antibiotic treatment and the extent of renal toxicity. However, in contrast to four patients of the control group, no patient in the amifostine group developed such severe mucositis that artificial ventilation became necessary. Pretreatment of neuroblastoma cell lines for 30 minutes with amifostine and the free thiol(WR-1065) did not reduce the cytotoxic effects of melphalan, carboplatin and VP-16. Evidence was obtained that the uptake of the activated thiol could be achieved by a polyamine transporter. Taken together, the data do not support the use of amifostine in high dose chemotherapy of neuroblastoma prior to autologous stem cell transplantation. However, amifostine may be more effective in conventional neuroblastoma therapy where protection of bone marrow stem cells is necessary.

Growth inhibition of neuroblastoma cells by lovastatin and L-ascorbic acid is based on different mechanisms.

Hydroxymethyl-glutaryl-CoA-reductase (HMG-CoA-reductase), the key enzyme for cholesterol synthesis and essential for the synthesis of the precursor for p21ras farnesylation, was inhibited in neuroblastoma cells by lovastatin or L-ascorbic acid. Both compounds inhibited clonogenic colony formation of neuroblastoma cells in soft agar. However, while the addition of mevalonate, the product of HMG-CoA-reductase, circumvented the inhibition by lovastatin it had no reversing effect on the inhibition by L-ascorbic acid. The role of reactive oxygen compounds generated by the degradation of catecholamines, and the pro-oxidative effects of L-ascorbic acid are discussed as mechanisms of action of L-ascorbic acid.

Isotachophoretic determination of phosphate splitting from Amifostine and p-nitrophenyl phosphate in serum and neuroblastoma cells.

Amifostine [WR-2721; H2N-(CH2)3-NH-(CH2)2-S-PO3H2] is used as a protecting agent in the chemotherapy of neuroblastoma. It is supposed that Amifostine will be transformed into its active form, the free thiol (WR-1065), easier by normal cells than by tumour cells. Analytical capillary isotachophoresis was used to determine the dephosphorylation of Amifostine in serum and on neuroblastoma cells and peripheral blood cells. Furthermore, the biological effects of Amifostine and its free thiol, on cell proliferation of neuroblastoma cells were measured in combination with Carboplatin. It was found that neuroblastoma cells did not split phosphate less efficiently than normal peripheral blood cells. Furthermore, neither Amifostine (as expected) nor the free thiol (not expected according to the theory) were able to inhibit the effects of Carboplatin. Therefore, the current hypothesis concerning the mode of action of Amifostine must be questioned.

Presence of bleomycin-detectable free iron in the alveolar system of preterm infants.

Chronic lung disease (CLD) is a major cause of long term morbidity in preterm infants. Reactive oxygen species (ROS) play an important role in the pathogenesis of CLD. We show that a high percentage (63 to 83%) of the investigated bronchoalveolar secretions (BAS) of neonates contain bleomycin-detectable free iron concentrations (0. 04-0.124 nmol/micrograms SC, median range). Beside the presence of redox-active iron several iron-binding proteins like transferrin, ferritin and lactoferrin were determined in BAS. Comparison of protein distribution within the first three days of life showed slight differences between the group of preterm infants who developed CLD and the neonates who recovered from RDS. Because of the existence of free iron we suggest a higher risk of hydroxyl radical formation in the alveolar space. In an artificial system with addition of iron and hydrogen peroxide we were able to demonstrate OH-radical production in BAS by electron paramagnetic resonance (EPR). OH-radical formation by H2O2 and iron in buffer solution was slightly enhanced in the presence of BAS, indicating the absence of OH-radical-scavengers in BAS.

SiMa, a new neuroblastoma cell line combining poor prognostic cytogenetic markers with high adrenergic differentiation.

We describe the establishment and characterization of a new neuroblastoma (Nb) cell line, SiMa, carrying the major recurrent chromosome changes associated with poor prognosis Nb, including amplification of N-MYC by formation of double minutes (dmin), der(1)t(1;17)(p35;q12) and der(22)t(17;22)(q22;p13), and loss of chromosome 11, documented at both initiation and late passage. In contrast to these cytogenetic stigmata of poor prognosis, analysis of catecholamine synthesis by high pressure liquid chromatography (HPLC) measurement revealed an advanced degree of adrenergic differentiation with high rates of 3,4-Dihydroxyphenylalanine (DOPA), noradrenaline, homovanillic acid (HVA), and vanillylmandelic acid (VMA) production. Contrastingly advanced differentiation and poor prognostic genetic markers combine to render SiMa a unique instrument for investigating the pathology and therapy of Nb.

Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review).

Anthracycline-derivatives are frequently used chemotherapeutics in treatment of numerous human malignancies. Anthracyclines are known for their complex cytotoxic mechanism involving i) inhibition of enzymes such as topoisomerase II, RNA polymerase, cytochrome c oxidase and others; ii) intercalation into DNA; iii) chelation of iron and generation of reactive oxygen species (ROS); iv) induction of apoptosis. Here, mechanistic aspects for successful cytostasis and for side effects, e.g. cardiomyopathy, are discussed. We emphasize recent developments in anthracycline-mediated apoptosis and focus on a well known representative, doxorubicin (adriamycin, adriblastin). We reflect on the role of oxidative stress and interactions with intracellular signaling pathways.

Kinetics of redox interaction between substituted 1,4-benzoquinones and ascorbate under aerobic conditions: critical phenomena.

Redox cycling is believed to be the most general molecular mechanism of quinone (Q) cytotoxicity. Along with redox cycling induced by a reductase, a similar process is known to occur via electron transfer from ascorbate (AscH-) to Q with formation of a semiquinone radical (Q.-): (1) Q + AscH- (k1)--> Q.- + Asc.- + H+ (2) Q.- + O2 --> Q + O2.-. The net effect of reactions (1) and (2) provides for the catalytic oxidation of AscH-, with Q serving as a catalyst. In this work, the kinetics of oxygen consumption accompanying this process were studied with several substituted 1,4-benzoquinones (BQ) at 37 degrees C in phosphate buffer, pH 7.40, using the Clark electrode technique. The value of k1 determined from the initial rate of oxygen consumption was typically found to increase when the one-electron reduction potential E(Q/Q.-) shifted to more positive values. With Q, for which E(Q/Q.-) is less than -100 mV, the rate of oxygen uptake (R(OX)) was found to be directly correlated with the [Q][AscH-] value independent of the concentration of individual reagents, remaining constant for a long period. With mono- and dialkyl-substituted 1,4-BQs, for which E(Q/Q.-) is higher than -100 mV, significant deviations from the above simple kinetic regularities were observed. In particular, R(OX) decreased dramatically with time and critical phenomena (the existence of certain concentrations of Q and/or AscH- above or below which the catalytic oxidation of AscH- ceased completely after a non-stationary period of short duration) were observed. These abnormalities can be explained on the basis of the kinetic scheme which contains, in addition to reactions (1) and (2), several side reactions including that between Q.- and AscH-. Implications of critical phenomena discovered in this study for the problems of Q toxicity and vitamin C avitaminosis are discussed.

Fully reversible redox cycling of 2,6-dimethoxy-1,4-benzoquinone induced by ascorbate.

The kinetics of cyclic redox transformation of 2,6-dimethoxy-1, 4-benzoquinone (DMOBQ)--the well-known effective anticancer agent--induced by ascorbate (AscH-) were studied in phosphate buffer, pH 7.40, at 37 degreesC using the Clark electrode and ESR techniques. The process is due to the electron transfer from AscH- to quinone (Q): Q + AscH- --> Q*- + Asc.- + H+ (1), followed by semiquinone (Q.-) oxidation: Q.- + O2 --> Q + O2.- (2). DMOBQ, taken even at submicromolar concentrations, effectively catalyzed AscH- oxidation that manifested itself by intensive oxygen consumption and an increase in the steady-state concentration of the ascorbyl radical (Asc.-). The rate of oxygen consumption, ROX, was kept almost constant for a long time. ROX was found to be proportional to the [Q][AscH-] product and not dependent on the concentrations of the individual reagents. The rate constant for reaction (1) determined from ROX and [Asc.-] was as much as 380 +/- 40 and 280 +/- 30 M-1.sec-1, respectively. When DMOBQ was mixed with the corresponding hydroquinone, QH2, in oxygen-free buffer, the ESR signal of Q.- which formed due to the equilibrium Q + QH2 left and right arrow 2Q.- + 2H+ (3) was observed. The equilibrium constant K3 of (2.6 +/- 0.4).10-5 and the change in the reduction potential, DeltaE3 = E(Q/Q.-) - E(Q.-/QH2), of -280 mV were calculated from the steady-state concentration of Q.- at pH 7.4 and 37 degrees C. From combination of DeltaE3 determined in this study with E7(Q/Q.-) reported in the literature, a value of +190 mV was calculated for the standard second one-electron reduction potential E(Q*-/QH2). The latter is lower by 270-230 mV than that for all the studied 1, 4-hydroquinones. The very beneficial combination of E(Q/Q.-) and E(Q.-/QH2) was suggested to be the basic reason for the perfect work of DMOBQ as a redox cycling agent and its pronounced anticancer activity.

Ascorbic acid stimulates DOPA synthesis and tyrosine hydroxylase gene expression in the human neuroblastoma cell line SK-N-SH.

Ascorbic acid is well known to induce noradrenaline synthesis in sympathetic nervous cells. In a series of experiments we found that incubation of the neuroblastoma cell line SK-N-SH with ascorbic acid (100-500 microM) for 2 h results in a significantly enhanced synthesis of 3,4-dihydroxyphenylalanine (DOPA) and dopamine. Additionally, cDNA-polymerase chain reaction (cDNA-PCR) analysis of relative mRNA levels corresponding to the enzymes involved in catecholamine synthesis revealed a 3-fold increase of tyrosine hydroxylase gene expression after 5 days of incubation with ascorbic acid (200 microM), whereas expression of dopamine-beta-hydroxylase was found to be unaltered. In summary the data give evidence that ascorbic acid leads to enhanced DOPA production in SK-N-SH cells by two different mechanisms: at the metabolic level after short-term incubation and by increasing the tyrosine hydroxylase gene expression after long-term incubation. Based on these data we suppose that enhancement of DOPA synthesis by ascorbic acid may be useful in the treatment of early Parkinson's disease.

Ubiquinone-0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone) as effective catalyzer of ascorbate and epinephrine oxidation and damager of neuroblastoma cells.

The kinetics of ascorbate (AscH ) and epinephrine (EP) oxidation in the presence of 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ) were studied in 0.05 M phosphate buffer, pH 7.4, at 37 degrees C by using a Clark electrode and ESR techniques. UQ at nanomolar concentrations displayed a pronounced catalytic effect on AscH oxidation which exceeded that of all reported organic catalysts tested in this system. The process was accompanied by the intensive oxygen consumption and increase in the steady-state concentration of the ascorbyl radical Asc.-. The rate of oxygen consumption (R[OX]) was maximal at the moment of reagent mixing ((R[OX]0) and then reduced over a few minutes until a steady-state level ((R[OX])SS) was achieved. (R[OX])0 was found to be proportional to [UQ][AscH-] without regard to the concentrations of the individual reagents; (R[OX])SS was directly related to [UQ] at a given concentration of AscH-. The difference between (R[OX])0 and (R[OX])SS decreased as [AscH-] decreased. The presence of a lipid phase (sodium dodecylsulphate micelles) only moderately decreased UQ activity as a catalyst of AscH- oxidation. Adding micromolar concentrations of UQ induced the acceleration of EP autoxidation. The capability of UQ to catalyze the oxidation of EP exceeded by approximately 25 times that of adrenochrome, a quinoid product of EP oxidation. These catalytic properties of UQ allowed us to predict its pronounced cytotoxicity, especially in the presence of AscH- and to cells of the sympathetic nervous system which are rich in catecholamines. This possibility was confirmed by experiments with human neuroblastoma cells in culture. The capability of UQ to injure neuroblastoma cell line SK-N-SH exceeded that of well-known neurotoxic agents 6-hydroxydopamine and menadione.

Cytotoxic effect of immunoconjugate composed of glucose-oxidase coupled to an anti-ganglioside (GD2) antibody on spheroids.

As a new treatment protocol for neuroblastoma, the chimeric (human/mouse) antiganglioside GD2 antibody chl4.18 is being clinically tested. To improve the therapeutic effect of the antibody alone, we are currently investigating the cytotoxicity of glucose-oxidase coupled to the antibody chl4.18 on spheroids of the neuroblastoma cell line SK-N-LO. The cytotoxic effect of glucose-oxidase is achieved by the production of hydrogenperoxide (H2O2) and probably by the following reaction of H2O2 with iron to form hydrogen radicals (OH.). The cytotoxicity of glucose-oxidase was measured by two viability tests (MTT and WST 1). After a 4 hour treatment of the spheroids with the immunoconjugate, a reduction of viability to 50% (MTT-test) and 25% (WST 1-test), respectively, was obtained. The difference between the results of these two tests, might be explained by the different measurement protocols.

Iron bound to ferritin catalyzes ascorbate oxidation: effects of chelating agents.

Ferritin is the main intracellular iron storage protein. Ferritin iron may be released by many reducing agents including ascorbate. In this work we report ferritin to catalyze the oxidation of ascorbate. The kinetics of this process were studied in detail in phosphate buffer (pH 7.40), at 37 degrees C by using the Clark electrode technique and ESR. The catalytic effect of ferritin manifested itself as the increase both in the rate of oxygen uptake and steady-state concentration of the ascorbate radical. The ferritin catalytic activity was found to be modified by iron chelators, EDTA. Desferal (DFO) as well as by ferrozine (FRZ) which is widely used in kinetic studies on ferritin iron release thanks to the formation of a coloured complex with Fe(II). While EDTA promotes the catalytic action of ferritin, DFO and FRZ diminished it. From the comparison of the kinetics of ascorbate oxidation obtained in the current work and data on the kinetics of ferritin iron release reported by Boyer and McCleary ((1987) Free Rad. Biol. Med. 3, 389-395), we conclude that iron bound to ferritin rather than the iron released is likely responsible for ferritin catalytic action. In addition, it has been concluded that the use of FRZ as an analytical reagent in kinetic studies of reductive ferritin iron release requires taking into account the competitive character of the formation of the Fe(II)-FRZ complex.