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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2975-2982
RED CELLS
The assessment of serum nontransferrin-bound iron in chelation
therapy and iron supplementation
William Breuer,
Aharon Ronson,
Itzchak N. Slotki,
Ayala Abramov,
Chaim Hershko, and
Z.
Ioav Cabantchik
Department of Biological Chemistry, Institute of Life Sciences,
Hebrew University of Jerusalem, Jerusalem, Israel; Department of
Internal Medicine and Nephrology Unit, Department of Pediatrics, Shaare
Zedek Medical Center, Jerusalem, Israel.
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Abstract |
Nontransferrin-bound iron (NTBI) appears in the serum of individuals
with iron overload and in a variety of other pathologic conditions.
Because NTBI constitutes a labile form of iron, it might underlie some
of the biologic damage associated with iron overload. We have developed
a simple method for NTBI determination, which operates in a 96-well
enzyme-linked immunosorbent assay format with sensitivity comparable to
that of previous assays. A weak ligand, oxalic acid, mobilizes the NTBI
and mediates its transfer to the iron chelator deferoxamine (DFO)
immobilized on the plate. The amount of DFO-bound iron, originating
from NTBI, is quantitatively revealed in a fluorescence plate reader by
the fluorescent metallosensor calcein. No NTBI is found in normal sera
because transferrin-bound iron is not detected in the assay. Thalassemic sera contained NTBI in 80% of the cases (range, 0.9-12.8 µmol/L). In patients given intravenous infusions of DFO, NTBI initially became undetectable due to the presence of DFO in the sera,
but reappeared in 55% of the cases within an hour of cessation of the
DFO infusion. This apparent rebound was attributable to the loss of DFO
from the circulation and the possibility that a major portion of NTBI
was not mobilized by DFO. NTBI was also found in patients with
end-stage renal disease who were treated for anemia with intravenous
iron supplements and in patients with hereditary hemochromatosis, at
respective frequencies of 22% and 69%. The availability of a simple
assay for monitoring NTBI could provide a useful index of iron status
during chelation and supplementation treatments.
(Blood. 2000;95:2975-2982)
© 2000 by The American Society of Hematology.
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Introduction |
The presence of nontransferrin-bound iron (NTBI) in the
circulation is a pathologic phenomenon, which generally occurs in patients with iron-overload conditions1-3 and is thought to
be absent from healthy individuals in whom virtually all the serum iron
is bound to transferrin (Tf). Until recently, NTBI has been thought
to arise from either repeated transfusions, which are required by patients with various hemolytic diseases,
hemoglobinopathies such as thalassemia, or excessive iron
absorption associated with hereditary hemochromatosis (HHC).
However, NTBI can also appear in the circulation of patients
undergoing chemotherapy,4,5 heart bypass
operations,6 and other conditions in which large amounts of
iron from hemoglobin catabolism are suddenly released into the
circulation. Recently, NTBI was also found in dialysis patients treated
for anemia with erythropoietin and iron supplements (personal
communication, Prof J.J.M. Marx, University Hospital, Uttrecht, Holland).
The clinical significance of NTBI is threefold: it is a potential
target of iron chelators; it could serve as an indicator of the iron
status of an individual who is already iron overloaded or at risk; and
it may participate as one of the causative factors in the tissue
loading with iron, which can lead to cardiac, hepatic, and endocrine
dysfunction. The first two points highlight the potential importance of
a convenient and reliable method for the detection of NTBI. In the
first case, it would allow the physician to assess the efficacy of an
iron-overloaded patient's chelation regimen and modify it accordingly.
In the second case, it could be used to help diagnose individuals who
already have or are predisposed to iron overload. This paper introduces
a novel assay for NTBI and shows its application to monitoring chelator
activity in patients with thalassemia and detecting NTBI in a high-risk
population such as patients with HHC and a lower risk population such
as patients on dialysis who receive iron supplementation. Results also
show, in accordance with previous reports,5 that NTBI can
occur in sera with less than full transferrin saturation.
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Materials and methods |
NTBI assay
The assay for NTBI is schematized in Figure
1.
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| Fig 1.
Scheme of the NTBI assay.
The scheme depicts the 4 basic steps of the assay for both normal
(left) and NTBI-containing (right) sera. The iron is depicted as a full
circle and the transferrin molecules denoted by T. Step 1: A serum
sample, which has been premixed with 100 mmol/L oxalate, 2.5 mmol/L
MnCl2, and 20 mmol/L HEPES pH 7.4, is added to DFO-coated
wells. The mobilized NTBI binds to the DFO on the plastic during a
2-hour incubation. Step 2: Only the DFO-bound iron remains after the
wells are washed. Step 3: Calcein-Fe complex (CA-Fe) is added to the
wells. CA-Fe is nonfluorescent because of quenching of calcein by the
bound iron. Step 4: The fluorescence is measured after a 2-hour
incubation, during which the remaining available DFO molecules withdraw
iron from CA-Fe, causing the released calcein to become fluorescent.
For normal serum, a maximum level of fluorescence is attained,
whereas for NTBI-containing serum, fluorescence is relatively
lower. The fluorescence generated is inversely proportional to the
concentration of NTBI in the original sample.
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1. Samples of 20 µL of serum (defined as "input sample") are
placed in disposable plastic 96-well plates ("mixing plate"). All
samples are prepared in duplicate.
2. To each sample is added 230 µL of a solution containing 100 mmol/L sodium oxalate (BDH Chemicals Ltd, Poole, UK), 20 mmol/L HEPES
(Biological Industries, Kibbutz Beit Haemek, Israel), pH 7.4 containing 2.5 mmol/L MnCl2 (Aldrich Chemical Co,
Milwaukee, WI). The oxalate-HEPES solution and the MnCl2 (1 mol/L stock) are kept as frozen aliquots and mixed just before use. The
iron content of the sodium oxalate is less than 0.001% according to the manufacturer. We have determined the iron concentration in the 100 mmol/L sodium oxalate solution as 0.7 µmol/L, using the bathophenanthroline sulfonate method (data not shown).
3. From the diluted samples, 100 µL is transferred in duplicate to
96-well plates coated with deferoxamine (DFO). For preparation of
plates see below.
4. After 2 hours of incubation at 37°C, the plates are washed
twice with distilled water, once with 5 mmol/L EDTA (pH 8.0), then
twice with distilled water.
5. To each well is added 0.1 mL of a preformed calcein-iron complex
(CA-Fe) consisting of 600 nmol/L calcein (Sigma Chemical, St Louis,
MO), 540 nmol/L ferrous ammonium sulfate (FAS) in 20 mmol/L HEPES, 150 mmol/L NaCl, pH 7.3 (HBS), and the plate is incubated for 2 hours at
37°C.
6. The fluorescence in the wells is determined in a multiwell plate
reader (BMG Lab Technologies, Offenburg, Germany) with excitation/emission filters of 485/538 nm and gain of 25.
Preparation of DFO-coated plates
1. A solution is prepared containing hydroxyethyl-starch-DFO, 0.5 mg/mL (Biomedical Frontiers, Minneapolis, MN), in 10 mmol/L sodium
phosphate (dibasic), pH 8.6.
2. From this solution, 100 µL/well is added to 96-well polystyrene
plates (F96 Maxisorp; Nunc, Roskilde, Denmark) and the plates are
incubated at 4°C for 72 hours.
3. The plates are washed twice with distilled water and, after shaking
off all excess water, can be used immediately, or stored at 4°C for
at least 2 months. The degree of variability in DFO activity between
wells of a plate, as determined by using CA-Fe de-quenching in blank
plates, is less than 4%. The degree of variability between different
plates is less than 11%.
Calibration
Calibration is shown in Figure 2.
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| Fig 2.
Calibration of iron concentration versus fluorescence.
A series of concentrations of iron, ranging from 0 to 400 µmol/L
(described in "Materials and methods"), was prepared in HBS
buffer containing either BSA (circles) or apo-Tf (triangles). A 20-µL
sample from each solution ("input sample") was mixed with either
HBS containing 2 mmol/L NaHCO3 (empty symbols) or
oxalate-Mn reagent (filled symbols) (final volume, 250 µL). Aliquots
of 100 µL were transferred to DFO-coated plates and processed in the
NTBI assay as described in "Materials and methods." The
fluorescence was read after 2 hours of incubation with CA-Fe and
plotted semilogarithmically against the concentration of iron in the
original 20-µL input sample (A). The most sensitive region of the
calibration curve (0-12.5 µmol/L Fe) was plotted separately as a
linear graph (B). Bars indicate standard deviation of the mean of 4 individual samples.
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1. To 0.1 mL of 10 mmol/L FAS (freshly prepared; Sigma Chemical) in
distilled water in a polyethylene tube is added 0.1 mL of 70 mmol/L
nitrilotriacetate, sodium form, pH 7.0. After 5 minutes, 0.3 mL water
is added to give 2 mmol/L Fe+++.
2. A serial dilution of 1:1 in water, using 50 µL for each dilution
step, is performed for up to 12 steps in polyethylene tubes, giving 50 µL of iron at concentrations from 400 down to 0.2 µmol/L.
3. To each tube is added 0.2 mL bovine serum albumin (BSA, Fraction V;
Sigma Chemical), 10 mg/mL in HBS.
4. A sample of 20 µL is transferred to the "mixing plate" and
subsequently processed in the same way as serum samples.
Preparation of CA-Fe
1. A stock solution of the CA-Fe complex is produced by mixing 20 mmol/L FAS (prepared fresh in deionized water) with 40 µmol/L calcein
(Sigma Chemical) to give final concentrations of 40 µmol/L calcein
and 36 µmol/L iron (CA-Fe ratio is 1.0:0.9).
2. The mixture is incubated in the dark at 37°C for 30 minutes.
3. The CA-Fe complex is divided into aliquots and stored frozen. The
solution is stable for up to 3 months in the freezer, but thawed
solutions are not reused.
4. For use in the assay, the concentrated stock CA-Fe (40 µmol/L:36
µmol/L) is freshly diluted 1:67 in HBS 150 mmol/L NaCl, 20 mmol/L
HEPES, pH 7.3 to produce a final concentration of 0.6 µmol/L CA-Fe.
Modification for measurements of NTBI in sera with transferrin
subsaturation values
The previously described method was modified for sera with
transferrin saturation values in the range of 30% to 60%, to minimize apo-Tf-mediated sequestration of NTBI, which had been mobilized by the
oxalate-manganese reagent. The NTBI solubilizing reagent used in step 2 of the method was changed from 100 mmol/L oxalate, 20 mmol/L HEPES, 2.5 mmol/L MnCl2, pH 7.4, to 200 mmol/L oxalate, 20 mmol/L
HEPES, 20 mmol/L MnCl2, 1 µmol/L FeCl3, pH
7.4. The addition of FeCl3 (BDH Chemicals, Ltd), prepared
as a 1 mmol/L stock in 0.1 mol/L HCl from anhydrous powder, was
required for lowering the concentration of available apo-Tf. The
Mn++ concentration was increased from 10 to 20 mmol/L and
the oxalate concentration was doubled from 100 to 200 mmol/L to ensure
a high oxalate-Mn ratio and to preclude the possible formation of
oxalate-metal precipitates.
The basis for the use of increased Mn++ concentration was
empirical. A series of transition metals were tested at different concentrations for the capacity to enhance the detection of NTBI in
thalassemic sera. The rationale behind this approach was to find a
metal that would block both specific (ie, apo-Tf) and nonspecific iron-binding sites that might be present in serum samples. Among the
metals tested, Mn++ was the most suitable because it
enhanced the relative changes in fluorescence and did not interfere
with the assay. Subsequently, we found that Mn++
facilitates the binding of Fe+++ by DFO, in the presence of
high oxalate concentrations. This was determined by using the iron
probe N-(fluorescein-5-thiocarbamoyl) desferrioxamine (Fl-DFO;
Molecular Probes, Eugene, OR), whose fluorescence is quenched
stoichiometrically by Fe+++ under conditions that mimic
those in the assay. The rate of quenching of Fl-DFO (2.5 µmol/L) by
ferric ammonium citrate (20 µmol/L) was 5-fold slower in the presence
(t1/2 = 12.5 minutes) than in the absence of
200 mmol/L oxalate (t1/2 = 2.5 minutes), but was only
2.6-fold slower when 20 mmol/L MnCl2 was added to the
oxalate (t1/2 = 6.5 minutes). The effect of
Mn++ was concentration dependent and was maximal at 20 mmol/L (Breuer et al, unpublished data). Hence, we attribute the
enhancement of the sensitivity of the assay by Mn++ to its
facilitation of DFO binding of iron. Thalassemic sera, which generally
have very low iron-binding activity, showed detectable NTBI in the
presence of 2.5 mmol/L Mn++; however, NTBI was detectable
in sera with higher iron-binding activity (low transferrin saturation)
only when 20 mmol/L Mn++ was used.
The above modification results in a 20% decrease in the fluorescence
signals obtained in the reagent blanks (bovine serum albumin [BSA] 10 mg/mL in HBS) and iron standards and, therefore, requires the
establishment of an appropriate calibration curve using the modified
oxalate-Mn-Fe reagent. This curve (Figure 5) is parallel to the one
shown in Figure 2. To preclude the possibility of false-positive
values, each assay plate contains 4 reagent-blank wells with 20 µL
BSA 10 mg/mL in HBS. These 4 wells provide the zero NTBI value for each
particular plate and are used to adjust the values obtained in each
test plate to the iron standard curve, which is generated in a
different plate. This adjustment depends on the variability in DFO
content between plates, which fluctuates between 0% and 11%.
Patient treatments
The study included 12 patients, aged 10 to 16, all with
transfusional iron overload. Seven had thalassemia major, 3 had
thalassemia intermedia, and 2 had aplasia. All the drug treatments
carried out in this study were within the framework of their routine
therapeutic regimens.
The 9 patients undergoing DFO infusion kinetics arrived at the clinic
in the morning, having been instructed not to take any medications
before their arrival. A serum sample was taken (time 0') and,
immediately afterward, the patient began to receive an infusion of DFO.
The total DFO dose was usually 1 g; in a few cases it was only 0.5 g.
Duration of the infusion was between 30 and 60 minutes.
Sera routinely taken for ferritin estimation were used for serum
sampling for NTBI in patients with end-stage renal disease (ESRD).
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Results |
Configuration of the NTBI assay
The procedure of the NTBI assay (schematized in Figure 1) is based
on the binding of mobilized NTBI to DFO immobilized in 96-well plates.
The amount of DFO-bound iron is quantified by use of the metal probe
calcein complexed to iron, which becomes fluorescent following the
withdrawal of the iron by DFO.7-9 We found that to obtain
optimal detection with the immobilized DFO, it was necessary to
mobilize the NTBI using a charged low-molecular-weight chelator,
oxalate. Nitrilotriacetate, which is used as a mobilizing agent in the
assay developed by Singh et al,3 is not compatible with our
method, because it can remove iron from transferrin when present
together with DFO11 (Figure 2). Oxalate does not appear to
share this property with nitrilotriacetate in our system, although it
can still mobilize NTBI. In preliminary experiments with thalassemic sera, we found that the presence of oxalate was necessary; NTBI was
detected in only 3 of 15 cases when sera were tested without oxalate,
but in 12 of 15 cases when tested with oxalate (data not shown). This
finding suggests that NTBI may have 2 distinct chemical forms, the
first directly chelatable by DFO and the second that requires prior
mobilization by a charged low-molecular-weight chelator. Only the
results obtained with oxalate are reported here.
Calibration of the NTBI assay
The sensitivity of the assay was examined by preparing a series of
iron concentrations in HBS solution containing BSA, 10 mg/mL (Figure
2A). The titration curve performed with HBS-BSA shows that the
iron-binding capacity of the wells is saturated when the iron
concentration of the input sample (defined in "Materials and
methods") is 12.5 µmol/L. The calculated total iron-binding capacity of each well, based on this value, is 100 pmol. This was
confirmed by exposing the wells to increasing concentrations of CA-Fe
complex and determining the fluorescence after 2 hours of incubation.
The curve of fluorescence units (FUs) generated versus picomoles iron
added (in the form of CA-Fe) leveled off at 112 pmol Fe/well (data not
shown), indicating that saturation of the DFO sites in the well occurs
at this value. The titration curve shown in Figure 2A for samples with
BSA was altered by the presence of oxalate-Mn. We attribute the
decrease in slope to the action of oxalate as a competing ligand, which
causes an apparent change in the affinity of the DFO in the well. The
failure to reach the maximal level of fluorescence (15 000 U) in the
presence of oxalate is attributable to contaminating iron in different components (100 mmol/L oxalate contains 0.7 µmol/L Fe, see
"Materials and methods"). The useful range of the titration curve
carried out under standard assay conditions (ie, in the presence of
oxalate-Mn) is shown in Figure 2B, corresponding to iron concentrations
of 0 to 12.5 µmol/L in the input sample. The relationship between FUs
obtained and iron concentration is linear down to about 8 µmol/L,
with a lower limit of detection of 1 µmol/L. A standard curve of the
type shown in Figure 2B is routinely used for calibrating NTBI in serum samples.
To show that transferrin-bound iron is not detected by the assay, we
generated Tf-Fe by incubating a fixed concentration of apo-Tf with
increasing concentrations of iron and determining the iron
concentration in the samples by the assay (Figure 2A). At the
concentration of apo-Tf used (3.2 mg/mL), the total theoretical iron-binding capacity was approximately 80 µmol/L. In the presence of
oxalate-Mn, iron was not detectable at concentrations below 80 µmol/L. This indicates that transferrin-bound iron is spared by the
assay. We have found that oxalate-Mn appears to be slightly superior to
HBS-NaHCO3 at sparing transferrin-bound iron. In contrast, nitrilotriacetate (5 mmol/L) causes iron to be detectable at
concentrations less than 80 µmol/L, indicating removal of iron from transferrin.
Application of the assay to serum samples from
iron-overloaded patients
We initially examined the working range of the assay as a function
of the serum sample volume (Figure 3). For
most thalassemic patients, the highest sensitivity was obtained with
serum samples of 8 µL applied per well. Under these experimental
conditions, the amounts of iron detected were within the iron-binding
capacity of the wells. Two thalassemic sera and 1 control serum were
serially diluted in HBS containing 10 mg/mL BSA and equivalent sample
volumes (20 µL) from each dilution were processed as described in
"Materials and methods." A linear correlation was observed
between the volume of serum in the wells, the fluorescent signal
generated, and NTBI in pmol/well (obtained from the standard curve in
Figure 2B) for both thalassemic sera. In Figure 3, the NTBI values are
expressed in terms of pmol/well. To convert pmol/well to pmol/µL
serum (= µmol/L NTBI in serum), we corrected for the volumes and
dilutions made. Thus, for the input sample of 20 µL serum, only 8 µL was finally applied to each well. Hence, to obtain the actual NTBI concentration in the serum in µmol/L, the pmol/well value was multiplied by the factor 2.5 (= 20/8). In this way, we calculated the
NTBI concentrations of the 2 thalassemic sera to be 8.7 and 5.5 µmol/L. The control serum sample showed virtually no change in signal
with increasing input volume of the original serum sample. The
apparently negative values obtained for the control are attributed to
the sequestration of contaminant iron in the oxalate-Mn reagent by
apo-Tf. This effect of apo-Tf on samples with oxalate-Mn was also
observed in Figure 2, where samples containing undetectable iron showed
an apparent increase in the fluorescence generated with apo-Tf, but not
with BSA. As shown in Figure 4, the
contaminant iron is not a drawback, but in fact, an asset, because it
provides an indication of the iron-binding capacity of a given serum
sample.
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| Fig 3.
NTBI as a function of the volume of serum added to the
test wells.
Aliquots of serum from 2 thalassemics (N.A. and M.M.) and a healthy
individual (Control) were serially diluted in HBS containing 10 mg/mL
BSA, and assayed for NTBI as described in "Materials and methods"
and in Figure 2. Depicted on the abscissa is the volume of the aliquot
of original serum in the 100-µL sample applied to each well. The
fluorescence intensity obtained for each sample is indicated on the
right scale in terms of raw fluorescence units (descending scale). The
fluorescence units were converted to iron concentrations (IC, in
µmol/L), using the calibration curve depicted in Figure 2B and then
to pmol/well by multiplying IC by the volume of original serum in each
well. Negative values are due to chelation of contaminant iron in the
assay reagent by serum transferrin. Bars indicate standard deviation of
the mean of 4 individual samples.
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| Fig 4.
Monitoring the dynamics of chelation by DFO with the NTBI
assay.
Serum samples of 9 patients with thalassemia were taken for NTBI
measurements immediately before (0 minute) and at intervals of 30 minutes during and after infusion of DFO (0.5-1.0 g, IV). The duration
of the treatment was 30 to 60 minutes, as indicated in the graph. The
NTBI levels are shown in µmol/L as mean values of quadruplicates
(SD < 10%). Negative values indicate chelation of contaminant iron
present in reagents by serum components.
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Reliability of the NTBI measurements
To assess the day-to-day reproducibility of the assay, we chose 16 thalassemic serum samples taken from 5 different patients and tested
them on 3 different occasions, while freezing the sera in the
intervals. The deviation of the individual NTBI measurements varied
from 0.8% to 26%; the average deviation for all 16 sera was 8.3%. We
also tested 9 patients repetitively at monthly intervals (Table
1). Six of the patients showed relatively
steady NTBI levels with less than 25% variability among samples. The 3 patients who showed significant fluctuations of more than 50% had
lower NTBI values than the others. Two of them (S.N. and A.A.) have thalassemia intermedia; the third (S.P.) has thalassemia major, but is
under aggressive combined chelation therapy of defferiprone (L1) daily
and DFO once monthly. Therefore, we attribute the large fluctuations in
these cases to individual variability rather than to assay-based
errors.
Among 47 control sera from individuals without known iron-overload, no
NTBI positives were found (under the assay conditions using 100 mmol/L
oxalate, 2.5 mmol/L MnCl2), with values ranging from
 1.8 to 5.2, indicating that the test does not give
false-positive values.
Application of the test to monitoring the course of chelation
therapy
We investigated the possible usefulness of the assay in monitoring
the effectiveness of chelator treatment. Patients were sampled
periodically during the course of intravenous (IV) infusion of DFO and
their serum NTBI was measured (Figure 4). The NTBI became undetectable
immediately after DFO infusion in 8 of 9 cases and reached apparently
negative values. Cessation of DFO infusion (at around 60 minutes) was
followed by a gradual return toward higher apparent NTBI values. The
phenomenon of gradual NTBI rebound after DFO treatment has been
documented previously by Porter et al,13 using a different
NTBI assay, based on the mobilization of NTBI with nitrilotriacetate
and its detection with the chelator L1. The NTBI rebound effect would
be consistent with the facts that DFO has a short plasma
half-life11 and that the plasma iron turnover in
patients with thalassemia is 15-fold greater than
normal.1,2,13,14
It should be emphasized that our results do not necessarily indicate
the transient disappearance of NTBI from the serum during DFO infusion,
but rather the presence of excess free DFO in the serum, which competes
with the DFO immobilized on the assay plates for the soluble iron in
the sample. The negative values are obtained due to binding of the
contaminant iron in the oxalate-Mn reagent (see "Materials and
methods") by the free DFO in the serum sample, similar to the
binding by apo-Tf in control serum samples. Therefore, although the
chelator is present in the circulation, the present assay gives no
indication of NTBI status, but rather monitors the chelator activity
within a given serum sample. Because chelator kinetics can be
variable,11,14 the assay could be a valuable indicator of
chelator handling and activity in individual patients. This is
illustrated by the patient N.R., who was known to be severely iron
overloaded at the start of the treatment and, unlike the other
patients, had never previously been treated with chelators (Figure 4).
In this case, a slight increase rather than decrease in the apparent
NTBI was obtained, presumably because the patient's iron load was in
excess of the DFO infused. A subsequent infusion of DFO (1 week later)
gave NTBI kinetics similar to those of the other patients (not shown).
Detection of NTBI in patients with transferrin saturation of
30% to 60%
It has been shown previously that NTBI can occur concurrently with
transferrin saturation levels of less than 60% in patients with
perturbations in iron metabolism.5 This feature
necessitated a modification of the present method in an attempt to
overcome the sequestration by apo-Tf of iron that had been mobilized by oxalate. Normal sera contain 20 to 30 µmol/L apo-Tf, or 40 to 60 µmol/L iron-binding activity, which is in excess of the observed NTBI
concentrations (< 17 µmol/L). In preliminary experiments, using
1:1 mixtures of normal and thalassemic sera, we found that NTBI was not
detectable in these mixtures when the oxalate-Mn reagent used for
thalassemic sera (100 mmol/L oxalate, 2.5 mmol/L MnCl2) was
used. However, NTBI was detectable when the oxalate-Mn reagent was
modified to 200 mmol/L oxalate, 20 mmol/L MnCl2, 1 µmol/L
FeCl3 (see "Materials and methods"). Although these
maneuvers caused a decrease in the fluorescence signals obtained in the reagent blanks (BSA 10 mg/mL in HBS) and required the establishment of
new standard curves for the oxalate-Mn-Fe reagent, they significantly increased the sensitivity of the assay in sera with low transferrin saturation. It is unlikely that these measures contributed to the
generation of false-positive results, because the zero NTBI value in
each assay plate is based on the signal obtained with the reagent blank
solution in that particular plate (Figure
5). The presence of 1 µmol/L
FeCl3, in addition to the contaminant iron in the
oxalate-Mn reagent (see "Materials and methods"), gave rise to
apparently negative NTBI values in NTBI-free samples (from 2 to
16 µmol/L). Because these values generally correlated well
with the transferrin saturation, they are considered to reflect the
iron-binding capacity in the sera and, as such, do not detract from the
reliability of the assay. Because the measures mentioned above do not
completely block apo-Tf in sera with low transferrin saturation, this
aspect of the assay will necessitate further optimization, possibly by
using a Co+++-bicarbonate complex to block transferrin, as
described by Gosriwatana et al.22
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| Fig 5.
Calibration of iron concentration in the presence of
normal and pathologic sera.
A series of concentrations of iron, ranging from 0 to 50 µmol/L, was
prepared as described in "Materials and methods." A 20-µL
sample from each iron solution was mixed with 20 µL of either 10 mg/mL BSA in HBS buffer (BSA; empty triangles) or sera from a healthy
individual (Normal, empty squares), a patient with end-stage renal
disease (ESRD, filled squares), a thalassemic patient (Thalassemic;
filled triangles), and a hemochromatosis patient (HC; filled circles),
and incubated for 20 minutes. To each mixture was then added 210 µL
oxalate-Mn-Fe reagent (200 mmol/L Na-oxalate, 20 mmol/L
MnCl2, 1 µmol/L FeCl3) to give a final volume
of 250 µL; aliquots of 100 µL were transferred to DFO-coated plates
and processed as before. The fluorescence was read after 2 hours of
incubation with CA-Fe and plotted semilogarithmically against the
concentration of iron in the original 20-µL input sample.
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The detection of exogenous NTBI added to various serum types was
examined (Figure 5). Iron in the form of Fe-nitrilotriacetate was mixed
with the sera, and NTBI was assayed using the oxalate-Mn-Fe reagent.
Four different sera were examined, 1 from a healthy individual and 3 from patients. The respective NTBI contents of the normal, the ESRD,
thalassemic, and HHC sera were determined previously as 0, 6.2, 9.6, and 11.5 µmol/L. Parallel samples were prepared containing 10 mg/mL
BSA. The BSA curve is defined as representative of a sample with
neither NTBI nor iron-binding capacity and serves as the calibration
curve. Based on this curve, fluorescence of more than 6600 U indicates
an NTBI-negative sample, whereas fluorescence of less than 6600 U
indicates an NTBI-positive sample. As expected, the normal serum, with
no NTBI and a high iron-binding capacity, produced much higher
fluorescence than BSA, and NTBI was detectable only when the
concentration of added Fe-nitrilotriacetate was more than 30 µmol/L.
This may not reflect the full iron-binding capacity of the serum,
because the binding conditions were suboptimal due to the absence of
added HCO3. The NTBI-containing sera showed a gradual
increase in NTBI content (manifested as decreasing fluorescence) with
increasing Fe-nitrilotriacetate input, although the relationship between the input iron and the signal obtained was also influenced by
the initial NTBI contents and iron-binding capacities of the sera.
Using the modified assay, 3 groups of patients were tested (Table
2): controls, a heterogeneous group without
any known iron overload, some of whom were receiving iron supplements;
HHC, with various levels of iron overload; and ESRD, among whom
approximately 80% were receiving erythropoietin and IV iron
supplements (none of the patients had received IV iron in the week
before sampling). The respective frequencies of NTBI-positive patients
in the 3 groups were 4.3%, 69%, and 22%. The presence of NTBI in the
control group, who were assumed to be at low risk, was surprising. One of the 2 NTBI-positive individuals had a transferrin saturation of 75%
and, as was subsequently discovered, had taken a daily dose of oral
iron supplement shortly before serum sampling. The reason for the
presence of NTBI in the second control sample was not determined. Based
on this finding, it is conceivable that some NTBI-positive samples in
the other groups may also be due to a transient rise in serum iron,
which indicates that repeated measurements are mandatory for
establishing the persistent presence of NTBI.
| |
Discussion |
The occurrence of NTBI in the sera of thalassemic patients was
originally thought to arise exclusively in cases of severe iron
overload. It was reasoned that the iron-binding capacity of serum
transferrin was overwhelmed, leading to a spillover of iron to other
low-affinity ligands.1 This was consistent with the
relatively high (> 80%) transferrin saturation found in such patients. However, more recently, NTBI was also detected in the sera of
patients without full transferrin saturation,5 which indicates that a fraction of NTBI may exist in a form that is not
accessible to apo-Tf. Because NTBI has now been found to occur in a
variety of pathologic conditions in the absence of chronic iron
overload,4-6 we have sought a means to identify NTBI also in populations of patients who are at relatively lower risk of iron
overload. This paper describes a novel approach for assessing NTBI,
which led to the development of an assay that is both sensitive and
convenient for handling large numbers of samples. The assay was applied
to a variety of sera for monitoring chelation kinetics in patients with
thalassemia and for detecting NTBI in patients with ESRD.
There are presently 2 main approaches for determining NTBI in biologic
fluids. One approach is based on mobilization of NTBI with an anionic
ligand (EDTA, citrate, or nitrilotriacetate) followed by
ultrafiltration and quantitation of the filterable iron by either
established methods1 or by subsequent complexation with the
chelator L1 and separation/identification by high-performance liquid
chromatography (HPLC).3 An alternate approach uses the antibiotic bleomycin, which combines with NTBI, but apparently not with
iron bound to transferrin or other proteins, to form highly reactive
complexes that are quantified by the amount of DNA cleavage products
that they generate.15 Although both these methods have been
used successfully for some time, the present assay offers the
advantages of technical simplicity and low labor intensity, without
loss of sensitivity or generation of false-positive values. The NTBI
concentrations obtained with the present assay are roughly comparable
to those obtained previously, though slightly higher. The reported NTBI
values in thalassemic sera obtained by various methods are in the range
of 0 to 9 µmol/L,1,14 which is roughly similar to the
values found for thalassemic patients in this paper, 0 to 10.6 µmol/L.
The chemistry of NTBI is probably more complex than was originally
thought,1 because it might be comprised of a heterogeneous mixture of complexes whose composition might vary with the degree and
type of iron overload or iron supplementation. Evidently, understanding
its nature in different disease states is essential for preventing its
occurrence. Analysis of NTBI by HPLC and high-resolution nuclear
magnetic resonance indicated the presence of citrate-iron and ternary
citrate-acetate-iron complexes. Interestingly, the chelation of NTBI by
DFO appeared to be a very slow process, requiring a number of hours
even at 1 mmol/L concentration of the chelator.23 The
presence of NTBI in sera without full transferrin
saturation5 indicates that a fraction of the iron may exist
in a form that cannot be bound directly by apo-Tf. Similarly, if NTBI
is not fully accessible to chelators such as DFO, this may explain its apparent rebound following cessation of DFO infusion13
(Figure 4). This possibility is reinforced by the requirement for
NTBI-mobilizing agents such as nitriloacetate or oxalate in the assays
based on binding to L13 or DFO (present paper). Indeed, we
found that the presence of oxalate in our assay enabled the detection
of NTBI in 12 of 15 thalassemic patients as opposed to only in 3 of 15 patients in its absence. This finding further supports our view
regarding the heterogeneity of NTBI. Although the bleomycin-based NTBI
assay does not use a mobilizing agent,15 ascorbate, which
is a required component, most likely fulfills this function while
bleomycin acts as the iron-binding moiety. At present it is not clear
why DFO, L1, or apo-Tf fails to mobilize some forms of NTBI, whereas small, negatively charged molecules such as oxalate (this article), EDTA,1 citrate,1 or
nitrilotriacetate3 can mobilize and shuttle it to these
high-affinity ligands. Conceivably, the latter ligands fail to access
the presumed polynuclear forms of the metal in NTBI and form the
required multiple coordination points for stable binding. Thus,
iron-mobilizing agents or shuttles as supplementary tools for
elimination of NTBI by chelators could have an important function in
all those cases where iron chelators alone might have failed. This has
therapeutic implications both in terms of novel chelator design and for
supplementing current chelation regimens with agents targeted to
mobilize NTBI. Previously, we have implemented a similar
shuttle-chelator combination strategy in iron chelation protocols for
the treatment of malaria infections.16
As to the origin of NTBI, one may infer that it represents a fraction
of iron that entered the circulation, either directly from the site of
intestinal absorption or from macrophages. That fraction presumably
failed to be absorbed by apo-Tf and, hence, aggregated into insoluble
deposits or was adsorbed to negatively charged pockets of serum
proteins. It is also conceivable that some iron aggregates may have
been formed intracellularly, before their entry into the circulation,
whether by loss or extrusion from iron-overloaded cells as
hemosiderin-like complexes or as polymer-iron complexes used for iron
supplementation of patients.
A major advantage of the present NTBI assay is its potential usefulness
for conveniently screening large groups of patients. A limited example
of such an application is shown in Table 2. The presence of NTBI in
69% of the patients with HHC was expected and observed accordingly.
However, the finding of 4.3% NTBI-positive patients in the control
group was not anticipated. It should be emphasized that only 15 of
these samples were from healthy individuals, whereas the remaining 31 were from patients with a variety of non-iron-overload conditions, who
were referred for tests of transferrin saturation or serum ferritin. We
are aware of the possibility that NTBI might occur only transiently in
these patients, perhaps as an immediate and short-term result of iron
supplementation, and might not be obtained in successive samplings.
With respect to patients with ESRD, the 22% frequency of NTBI is
clearly significant, yet it may also represent a transient form of
NTBI. Studies are under way to determine the persistence of NTBI in
these patients over time and its correlation with iron supplementation
and with other diagnostic indicators of iron status (Slotki et al, in
preparation). We have also considered the possibility that patients
with ESRD may have elevated serum Al+++ levels, which could
masquerade as NTBI in our method, because DFO also binds
Al+++. This possibility is not likely for 2 reasons. First,
serum Al+++ levels rarely exceed 40 µg/L (= 1.5
µmol/L),17,18 which is considerably below the binding
capacity of apo-Tf (about 50 µmol/L), the major
Al+++-binding protein in the serum.19 Second,
the assay is relatively insensitive to Al+++ due to the
lower affinity of DFO for Al+++ than for Fe+++
(respective stability constants 1022 and 1032).
Nonetheless, we examined the question directly by premixing serum
samples from NTBI-positive and NTBI-negative ESRD patients with
increasing concentrations of AlCl3, and subjecting the
samples to the NTBI assay. No additional NTBI was detectable at
Al+++ concentrations up to 50 µmol/L in any of the sera.
The immediate potential applications of the assay presented here
include routine monitoring of NTBI status in iron overload, assessment
of chelator activity and handling during chelator therapy, and
screening of selected patient populations for NTBI. The screening application may be instrumental in uncovering the possible connection between NTBI and various potentially iron-associated pathologies, such
as cardiovascular disease.20,21
Note added in proof. The present assay was simplified by using
fluorescent-DFO analogs that undergo quenching upon stoichiometric binding of the metal (this work and also Breuer et al, manuscript submitted). Both the present and the modified assays can be applied either in the presence (indirect assay [IA]) or absence (direct assay
[DA]) of shuttle agents such as oxalate. The DA detected virtually no
NTBI in serum samples of normal individuals, whether or not they were
supplemented with more than 95% saturated Tf. The IA, however,
detected 1-5% iron mobilized from more than 60% iron saturated Tf.
This indicates that NTBI values obtained by the IA from samples with
relatively high (more than 60%) Tf saturation might include a
substantial component derived from Tf itself. Indeed, we found that
among 14  E Hb patients from Thailand (all with Tf saturations of
more than 95%) that were not under chelation treatment, 86% of them
had NTBI levels of 1 to 8 µM (mean 2.9 ± 1.7 µM) as detected by
the DA and 2.4 to 11 µM (mean 3.7 ± 2.7 µM) by the IA. In the
same type of patients orally chelated with L1 for up to 2 years, NTBI
values (µM) dropped significantly (P < 0.05, paired
t-test) from 3.98 ± 1.31 to 1.35 ± 0.48 by the DA and from
8.8 ± 0.3 to 7.1 ± 0.4 by the IA. The DA also detected NTBI (0.4 to
1.1 µM; 0.6 ± µM) in 9% of samples from primary hemochromatotic patients (n = 31; 92 samples), but in none of the ESRD patients (Israel). However, because most HC and ESRD sera had Tf saturations much less than 70%, the IA shown in this article provided a minimum estimate of their NTBI levels.
| |
Acknowledgments |
We would like to thank the staff and patients of the Children's Day
Hospital at Shaare Zedek Medical Center, Jerusalem, for their
cooperation; Ms Hava Glickstein for excellent technical assistance; and
the partners of the Biomed II group, Profs J. J. M. Marx, R. C. Hider, and P. Brissot, for advice and encouragement. The method was
filed for intellectual rights on December 17, 1998, application no. 127621.
| |
Footnotes |
Submitted June 17, 1999; accepted January 6, 2000.
Supported by a grant from the Israel Science Foundation and by Biomed
II Programme of the EEC.
Reprints: William Breuer, Department of Biological
Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction