Desferrioxamine-chelatable iron, a component of serum non–transferrin-bound iron, used for assessing chelation therapy by William Breuer, Marieke J. J. Ermers, Pensri Pootrakul, Ayala Abramov, Chaim Hershko, and Z. Ioav Cabantchik Blood Volume 97(3):792-798 February 1, 2001 ©2001 by American Society of Hematology

Scheme of the DCI assay.The scheme depicts the steps of the assay for both normal (left) and NTBI-containing sera (right). Scheme of the DCI assay.The scheme depicts the steps of the assay for both normal (left) and NTBI-containing sera (right). The iron is depicted as a filled circle and the Tf molecules denoted by ‘T’. Step 1: Serum samples are mixed with reagent A (HBS containing 2.5 μM fluorescein-DFO [Fl-DFO, *]) or reagent B (same as reagent A, but containing 100 μM DFO, **) in the wells. In reagent A the accessible Fe binds to the Fl-DFO and quenches its fluorescence, whereas in reagent B the Fe binds to the excess nonfluorescent DFO rather than to Fl-DFO. Step 2: Fluorescence is determined after a 2-hour incubation. In normal serum, the ratio of fluorescence of samples treated with reagent A and B is near 1, whereas in Fe-containing serum, the fluorescence in reagent A is lower than in B, giving a ratio less than 1. The ratio of the fluorescence readings (A/B) is inversely proportional to the concentration of DCI in the original sample. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

Calibration of Fe concentration versus fluorescence Calibration of Fe concentration versus fluorescence.A series of concentrations of Fe, ranging from 0 to 200 μM (described in “Patients, materials, and methods”), was prepared by serial dilution of Fe:NTA in HBS buffer (input sample). Calibration of Fe concentration versus fluorescence.A series of concentrations of Fe, ranging from 0 to 200 μM (described in “Patients, materials, and methods”), was prepared by serial dilution of Fe:NTA in HBS buffer (input sample). From each dilution 20 μL was transferred to 96-well plates followed by 100 μL 2.5 μM Fl-DFO in HBS. Replicate wells were treated with the same reagents containing in addition 100 μM DFO, to give the “maximal” fluorescence for each sample. The fluorescence ratio (expressed as “% of maximal value” obtained in the presence of excess DFO) was calculated for each sample (see “Patients, materials, and methods”) and plotted semilogarithmically against the concentration of Fe in the original 20-μL input sample (A). The linear region of the calibration curve (0-6.25 μM Fe) is shown in panel B. The values are averages ± SD of 6 separate calibration curves. The boxed area labeled “48 control sera” represents the range of values obtained for serum samples of 48 individuals without Fe overload. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

DCI in patients during L1 therapy DCI in patients during L1 therapy.Blood samples from 11 patients with thalassemia major were taken immediately before (0 minute) and at intervals of 30 minutes after administration of L1 (75 mg/kg) per os. DCI in patients during L1 therapy.Blood samples from 11 patients with thalassemia major were taken immediately before (0 minute) and at intervals of 30 minutes after administration of L1 (75 mg/kg) per os. Two groups of patients were tested, 8 in Israel (open symbols) and 3 in Thailand (filled symbols). Serum was isolated from the samples after overnight storage at 4°C, and stored frozen. All samples were assayed for DCI in duplicate as described in “Patients, materials, and methods.” Dashed line indicates 0 value. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

Spectrophotometric determination of Fe transfer from L1 to DFO Spectrophotometric determination of Fe transfer from L1 to DFO.A complex of L1-Fe was formed by mixing Fe and NTA (5 mM FAS:35 mM NTA) with L1 to give final concentrations of 100 μM L1 and 10 μM Fe, followed by incubation for 1 hour at room temperature. Spectrophotometric determination of Fe transfer from L1 to DFO.A complex of L1-Fe was formed by mixing Fe and NTA (5 mM FAS:35 mM NTA) with L1 to give final concentrations of 100 μM L1 and 10 μM Fe, followed by incubation for 1 hour at room temperature. A spectrum (range, 350-550 nm) of the L1-Fe complex was obtained (“L1-Fe complex”), followed by addition of 50 μM DFO to the cuvette, incubation for 10 minutes, and determination of a second spectrum (“L1-Fe complex + DFO”). The “DFO-Fe complex” was generated by mixing Fe and NTA (5 mM FAS:35 mM NTA) with DFO, to give final concentrations of 50 μM DFO and 10 μM Fe and incubating for 1 hour. The absorbance dip at 470 nm is due to instrument noise. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

Time dependence of Fe transfer from L1 to Fl-DFO Time dependence of Fe transfer from L1 to Fl-DFO.Preformed L1-Fe complexes containing 2 μM Fe and increasing concentrations of L1 (0-16 μM) were prepared from Fe:NTA and L1 as described in Figure 4. Time dependence of Fe transfer from L1 to Fl-DFO.Preformed L1-Fe complexes containing 2 μM Fe and increasing concentrations of L1 (0-16 μM) were prepared from Fe:NTA and L1 as described in Figure 4. At 0 minute, 20 μL of each solution was mixed with 100 μL 2.5 μM Fl-DFO in HBS, and the fluorescence was monitored with time. The final concentrations of both Fe and Fl-DFO were 2 μM in each system, whereas the concentration of L1 (μM) varied: ▪, 0; ●, 2; ▴, 4; ▾, 8; ♦, 16. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

Time dependence of Fe transfer from L1 to apo-Tf Time dependence of Fe transfer from L1 to apo-Tf.Two solutions were prepared in HBS, one containing 12 μM Fe as Fe:NTA, and the other preformed L1-Fe complexes containing 100 μM L1 and 12 μM Fe. From each solution 20 μL was mixed with an equal volume of eit... Time dependence of Fe transfer from L1 to apo-Tf.Two solutions were prepared in HBS, one containing 12 μM Fe as Fe:NTA, and the other preformed L1-Fe complexes containing 100 μM L1 and 12 μM Fe. From each solution 20 μL was mixed with an equal volume of either HBS or HBS containing 25 μM apo-Tf, and incubated for 15 minutes at room temperature. To each mixture was added 100 μL 2.5 μM Fl-DFO in HBS and the fluorescence was monitored with time: ▪, Fe + apo-Tf; ●, L1-Fe + apo-Tf; ▾, L1-Fe; ▴, Fe alone. The final concentrations of L1, Fe, apo-Tf, and Fl-DFO in the reaction mixture were 14.3, 1.7, 3.6, and 1.8 μM, respectively. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

Apo-Tf extracts Fe from L1-Fe complexes formed in vivo Apo-Tf extracts Fe from L1-Fe complexes formed in vivo.Serum samples taken from 2 patients (NN and AH) immediately after L1 administration (▪) (Figure 3) were mixed with apo-Tf (●) or DFO (▴) (final concentrations 0.5 mg/mL and 50 μM, respectively) for 15 m... Apo-Tf extracts Fe from L1-Fe complexes formed in vivo.Serum samples taken from 2 patients (NN and AH) immediately after L1 administration (▪) (Figure 3) were mixed with apo-Tf (●) or DFO (▴) (final concentrations 0.5 mg/mL and 50 μM, respectively) for 15 minutes at room temperature. The samples were then assayed for DCI as described. Dashed line indicates 0 value. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology

DCI in patients during L1-DFO combination therapy DCI in patients during L1-DFO combination therapy.Blood samples of 8 patients with thalassemia major were taken immediately before (0 minutes) and at intervals of 30 minutes after oral administration of L1, followed by 30- to 60-minute intravenous infusion ... DCI in patients during L1-DFO combination therapy.Blood samples of 8 patients with thalassemia major were taken immediately before (0 minutes) and at intervals of 30 minutes after oral administration of L1, followed by 30- to 60-minute intravenous infusion of DFO (shown by arrows). Serum was obtained from the blood samples after overnight storage at 4°C and subsequently stored frozen. All samples were assayed for DCI in duplicate as described in “Patients, materials, and methods.” Dashed line indicates 0 value. William Breuer et al. Blood 2001;97:792-798 ©2001 by American Society of Hematology