Summary. Hydroxyl radical formation catalysed by nontransferrin-bound iron (NTBI) might contribute to transplantation-related complications. The occurrence of NTBI in 10 adult allogeneic stem cell transplantation (SCT) patients was followed for 20 d. The transferrin saturation reached 99% on d 24 and remained . 80% thereafter. NTBI, measured as bleomycin-detectable iron, was detected for 6±18 d in all patients with a peak on d 24. High transferrin saturation levels were associated with the appearance of NTBI with a threshold at 80% saturation. Prevention of the potential deleterious effects of NTBI might reduce transplantation-related morbidity.Keywords: NTBI, iron, transferrin saturation, stem cell transplantation, bleomycin-detectable iron.Erythropoiesis, the main route of iron utilization, is temporarily halted by the myeloablative conditioning given before stem cell transplantation (SCT). The toxicity of the conditioning often affects not only haematopoiesis but also other organs, including the liver from which stored iron may be liberated as a result of hepatocellular injury (McDonald et al, 1986). Liver damage may also disturb transferrin synthesis. Thus, increasing saturation of serum transferrin results both from iron accumulation and a decline in the transferrin concentration, which, together, may lead to the appearance of potentially toxic nontransferrin-bound iron (NTBI) in the circulation. PATIENTS AND METHODSTen consecutive adult patients undergoing allogeneic SCT with cyclophosphamide/total body irradiation (CY/TBI) were enrolled in the study. The conditioning consisted of CY 60 mg/kg/d on d 26 and 25 (in relation to the SCT on d 0), and TBI 12 Grays fractionated in six doses from d 24 to d 0. The immunosuppressive prophylaxis consisted of i.v. cyclosporin, a short course of methotrexate and oral methylprednisolone (Ruutu et al, 2000).The baseline samples were collected on d 210 to 26, daily samples until d 114. The transferrin saturation was calculated using the formula: serum iron (mmol/l)/serum transferrin (g/l) Â 3´98. NTBI in serum was determined using the bleomycin-detectable iron (BDI) assay (Evans & Halliwell, 1994) modified for small serum volumes. The samples were measured in parallel with a corresponding blank without the addition of bleomycin. The absorbance value of the blank was deducted from that of each sample. The reagent blank value was deducted from the absorbance values of the standards, and a standard curve between 0´1 and 3 mmol/l was calculated by linear regression from each series. According to a validation study (unpublished observations), samples with BDI levels $ 0´1 mmol/l were considered positive for NTBI. RESULTSAt baseline, the mean serum total iron and mean transferrin levels were within the reference ranges. The mean transferrin saturation was 56%, but the variation was wide (Fig 1). NTBI was present in three of the nine evaluable patients; they also had transferrin saturation . 80%.The mean serum transferrin level decreased throughout the study period. During the f...

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Summary. Myeloablative treatment results in iron accumulation and the appearance of non-transferrin-bound iron (NTBI) in the circulation, which may contribute to treatment-related organ damage and susceptibility to infections. The aim of this study was to investigate the efficacy of human apotransferrin in the binding of NTBI in patients receiving an allogeneic stem cell transplant after myeloablative conditioning. A single intravenous 100 mg/kg dose of apotransferrin was given to six adult patients on d 3 after the transplantation. Initially, all patients had serum transferrin saturation above 80% and NTBI in their serum. After the apotransferrin injection, serum NTBI became undetectable in all patients and transferrin saturation decreased to 30-50%. Serum transferrin increased by an average of 1AE95 g/l. The administered apotransferrin was subsequently converted into monoferric and diferric transferrin forms. NTBI reappeared and transferrin saturation again exceeded 80% 12-48 h after the injection in four patients and after 6 d in one patient. NTBI remained non-detectable for the whole 12 d follow-up period in one patient. The apotransferrin injection was well tolerated and no adverse events with probable association with the apotransferrin were observed. Repeated apotransferrin infusions might completely eliminate NTBI and iron-induced toxicity during myeloablative therapy.

Background: A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method. Methods: The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis. Results: The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 μmol/L) and a high (1.5 μmol/L) control, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 μmol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay. Conclusions: The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.

Free iron induced hydroxyl radical formation is one possible mechanism for tissue injury during cytotoxic therapy. We studied the appearance of free, non-transferrin-bound iron (NTBI) at baseline and during the 20-d period after the onset of cytotoxic chemotherapy in patients with haematological malignancy undergoing intensive chemotherapy or conditioning for autologous stem cell transplantation (aSCT). NTBI was detected on average for 15.6 d in patients treated with chemotherapy only, and for 6.1 d in patients undergoing aSCT. The recovery of the bone marrow function coincided with the disappearance of NTBI. The type of the conditioning regimen was also associated with the appearance of NTBI. The timing of the presence of NTBI accords with the presence of the most important non-infectious complication of intensive chemotherapy and autologous transplantation, mucosal injury, and free iron is likely to contribute to this and probably other complications of the intensive treatments.