ANALYTICAL

BIOCHEMIS~Y

186,320-323

(1990)

A Direct Method for Quantification of Non-transferrin-Bound Iron Surinder Singh,* Robert C. Hider,* and John B. PorterP *Department of Pharmacy, King’s College London (KQC), University of London, Manresa Road, Chelsea, London S W3 6LX, United Kingdom, and TDepartment of Haematology, University College Hospital and Middlesex School of Medicine, 98 Chenies Mews, London, WClE 6HX, United Kingdom

Received

November

20,1989

A direct method for quantification of non-transferrin-bound iron has been developed. This assay relies on the use of a large excess of a low affinity ligand (nitrilotriacetic acid, NTA) which removes and complexes all low molecular weight iron and iron nonspecifically bound to serum proteins. Iron bound to transferrin, ferritin, desferrioxamine, and its metabolites is unaffected. The Fe-NTA complex present in the serum ultrafiltrate is then quantified using an automated HPLC procedure where on-column derivatization with a high affinity iron chelator (3-hydroxy-1-propyl-a-methylpyridin-4-one) takes place. The iron complexes of desferrioxamine and its metabolites are unaffected by the above-derivatization procedure. With minor modifications, this method is equally applicable for the quantification of low molecular weight iron in other biological fluids. Q 1990 Academic Press, Inc.

Patients may become iron overloaded either from repeated blood transfusions or from increased iron absorption. In man there is no effective excretory mechanism for excess iron and as this metal accumulates transferrin becomes saturated and inevitably an appreciable proportion of iron released by the reticuloendothelial system (RES)l (1) is unable to bind to transferrin. The precise nature of this form of iron (non-transferrin-bound iron, NTBI) is not known, but is thought to consist of iron complexed to physiological ligands and proteins present in serum (2). More recently it has been suggested that NTBI is almost exclusively present as 1 Abbreviations used: REX, reticuloendothelial system; 3-hydroxy-lpropyl-2-methyl-pyridin-4-one; (CP22),-Fe, tris+hydroxy-1-propyl-2methyl-pyridin-4-one-iron complex; DFO, desferrioxamine; FO, ferrioxamine; NTA, nitrilotriacetic acid; NTBI, non-transfetin-bound iron; Mops, morpholinapropanesulfonic acid. 320

iron citrate (3). This however is unlikely as the free citrate concentration in serum (due to the formation of calcium and ma~esium complexes) is limited to 1W5 M (4) and at pH 7.4, only 5 X lo-’ M iron(II1) can be solubilized as FeLH- (L = citrate) (5). Iron above this concentration will lead to the formation of large polynuclear species. Studies from our laboratory indicate that the high concentration of proteins present in serum provides ample nucleation sites for iron oligomer formation {e.g., carboxylate groups on albumin) and these result in the formation of predominantly small protein-associated polynuclear clusters which are readily chelatable (6). Although quantitatively small at any one time, there is considerable flux through this compartment due to the breakdown of red blood cells and release of iron through the RES. NTBI has been shown to be taken up rapidly by organs such as the liver and the heart (7-Q). This uptake is many times faster than that of transferrin-bound iron. Uptake of NTBI by the liver is thought to involve a translocation process dependant on membrane potential (10). NTBI is capable of generating free radicals which can initiate membrane damage and this possibly accounts for the hepatic and myocardial damage associated with iron overload. For this reason it is important to monitor and accurately quantify this potentially toxic iron fraction. For the analysis of NTBI in clinical situations, a rapid, simple, and reproducible assay with scope for easy automation is required. A number of methods are currently available for NTBI quantification (11-14). However many are based on simple calorimetric quantification which is insu~ciently sensitive for measuring low levels of NTBI. They are also influenced by the presence of perturbing chromophores, for instance ferrioxiamine (FO) and iron-complexed metabolites of DFO present in serum of patients undergoing chelation therapy. In some ~03.2697/90

$3.00

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

NON-T~NSFERRIN-BOUND

assays, EDTA (11) is used to remove iron nonspecifically bound to serum proteins. Unfortunately the high concentrations involved are capable of removing small amounts of iron bound to transferrin, FO, and iron complexes of DFO metabolites. In other assays nonspecific protein-bound iron is not quantified (13,14). The major problem however is the poor reproducibility of some of these methods. This is especially true for indirect methods which are based on the production and quantification of free radical degradation products. Such assays are likely to be influenced by the oxidant/anti-oxidant status of serum and the presence of other redox active metals such as copper. None of the methods developed so far are ideally suited to NTBI quantification in ironoverloaded sera. MATERIALS

AND

METHODS

IRON

321

QUANTIFICATION

1fXP

-.-

-/A--

go-

r)

-

-

-.

.

80-

8' .'

'FI 'O-/ L .

0 601 E e 50 d 4oL * 302dlo-

@O

10

I

20

,

30

I

40

SO

I

60

70

t

I

80

90

160

NTA (mM)

FIG. 1.

Removal of iron (30 PM) nonspecifically bound (8% solution prepared in serum ultrafiltrate) at various tions of NTA.

to albumin concentra-

Chemicals

DFO was obtained from Ciba-Geigy (Horsham, UK); NTA (disodium salt), ferric nitrate (nanohydrate), and morpholinopropanesulfonic acid (Mops) were from Aldrich; bovine serum albumin was from Sigma; HPLC grade acetonitrile was from BDH (Poole, UK). 3Hydroxy-1-propyl-2-methyl-pyridin-4-one (CP22) was synthesized as previously described (15) (available on request from the authors). Milli-Q water was used throughout the study. Removal of Iron Nonspecifically Bound to Albumin To an 8% bovine serum albumin solution prepared in serum ultrafiltrate was added 5gFe(5 &i) as iron malate (final concentration 30 PM Fe:60 PM malate). The solution was then allowed to equilibrate for 24 h at 25°C. Varying concentrations of NTA were then added and ultrafiltered using Amicon centriflo-CF 25 (MW 25,000 cutoff) with an applied centrifugal force of 1OOOg(Denley BR-401, UK) for 20 min. The 5sFepresent in the ultrafiltrate (which is representative of dissociated iron) was then counted using an LKB-Wallac 1282 compugamma CS gamma counter. Quantification of NTBI Serum samples obtained from patients undergoing DFO infusion were stored frozen (within 1 h of collection at -20°C) until time of analysis. No significant difference in NTBI levels was observed when either serum or plasma was used. To 0.9 ml of serum was added 0.1 ml of 800 mM NTA (pH 7) and allowed to stand for 15 min at 25°C. The solution was then ultrafiltered using Amicon centriflo-CF 25 filters. The ultrafiltrate (20 111)was then injected directly onto the HPLC system. A MiltonRoy (Stone, UK) CM 4000 HPLC system complete with a uv-visible detector (Spectra-Monitor 3100) and integrator (CI 4100) was used in the study. A Chrompak

(Chrom-Spher-ODS, 5 pm, 10 cm X 3 mm) glass column was used. Chromatographic conditions were as follows: flow rate, 0.8 ml/min; mobile phase, isocratic containing 20% acetonitrile and 3 mM CP22 in 5 mM Mops, pH 7; visible detection, 450 nm. A standard curve was generated by injecting varying concentrations of iron prepared in a 100-fold excess of NTA. RESULTS

AND

DISCUSSION

The method developed for quantification of NTBI should only measure potentially toxic iron fractions such as low molecular weight iron and iron nonspecifically bound to serum proteins. Muted

Development

An iron chelator is required to remove iron nonspecifically bound to serum proteins. The chelator used however should be carefully selected in view of the problems associated with EDTA. Ideally the chelator should have sufficient binding affinity to remove all iron nonspecifically bound to proteins present in serum but which does not compete for iron bound to transferrin, serum ferritin, DFO, or its metabolites. A ligand which fulfills these requirements is NTA. NTA, at all the concentrations studied (O-100 mM, results not shown), does not compete for transferrin-bound iron. Indeed, NTA is often used to load transferrin with iron. We have previously demonstrated that NTA does not compete for iron bound to DFO even at a lOOO-fold excess (16). The ability of NTA to remove nonspecifically bound iron from albumin is shown in Fig. 1. At a final concentration of 80 mM NTA > 98% iron removal was achieved. As work by Pape et al. (17) indicates that NTA-catalyzed removal of iron from ferritin is an extremely slow process, requiring hundreds of hours to remove appreciable quantities, it is unlikely that NTA will mobilize appre-

322

SINGH,

HIDER,

AND

PORTER

D

lo&M

FIG. 2. Methods

HPLC chromatogram for chromatographic

of (A) 10 fiM Fe-(CP2213, conditions.

Fe- NTA

(B) 10 pM Fe-NTA,

ciable amounts of iron during the short period required for handling of samples. Contributions from this source are therefore negligible. Thus the net effect of adding excess NTA to serum will result in the removal and complexation of all iron nonspecifically bound to serum proteins and low molecular weight iron bound to physiological ligands. Thus all the iron in the fraction of interest is quantitatively converted to the Fe-NTA complex. The solution is then subjected to ultrafiltration which separates the serum proteins from the low molecular weight fraction including Fe-NTA and FO.

HPLC

Separation

and Quantification

Deionized

(Cl deionized

25/4M

Water

water,

FO

and (D) 25 pM FO. Refer

to Materials

and

2 mM in the mobile phase. No difference in retention time or peak shape was observed when either a preprepared (CP22),-Fe complex or the Fe-NTA was injected on the column (Fig. 2). This is largely due to the high binding affinity of CP22 for iron relative to NTA and the high kinetic lability of both ligands. In contrast the hexadentate FO due to its slow kinetic lability is unaffected by the on-column derivatization procedure. The presence of CP22 in the mobile phase also serves to remove iron contamination in the buffer and the column in addition to ensuring that the (CP22)3-Fe complex remains intact under chromatographic conditions. To fur-

of NTBI

The serum ultrafiltrate containing iron-NTA and FO must be separated and quantified. For improved sensitivity and selectivity, an HPLC-based method was chosen. The use of a flow cell allows measurements of only the desired species without contribution from perturbing chromophores which would otherwise be present. In the first step of the quantification process, iron bound to NTA is converted to form the colored ((X22),-Fe complex (Lax 450 nm, E= 5500 M-l cm-‘) which is quantified in this assay. CP22 was chosen due to its high binding affinity for iron and favorable retention properties on reversed-phase HPLC columns. To prevent the possibility of iron removal from FO by CP22, on-column derivatization was selected. This procedure is especially useful for samples that are not analyzed immediately. Complete complexation of Fe-NTA to form (CP22&-Fe takes place immediately (4 s) if CP22 is present at or above

NTBI

(@A)

FIG. 3. Standard curve of peak height against iron concentration obtained on injection of various concentrations of Fe-NTA onto the HPLC system. Each point represents the mean of three separate measurements.

NON-TRANSFERRIN-BOUND 6r

IRON

323

QUANTIFICATION

therapy in a variety of iron-overloaded disorders. Figure 4 shows a set of serum NTBI values obtained from a patient undergoing high dose (100 mg/kg/day) intravenous DFO infusion. A large drop in the NTBI levels was observed with the onset of chelation therapy. With continual infusion of the drug, NTBI values eventually approach the base level and occasionally drop below zero, indicating incompletely saturated transferrin levels. We are currently making systematic investigations into the various relationships among NTBI, chelation therapy, and DFO metabolism. Days

FIG. 4.

Serum NTBI levels obtained from an iron-overloaded patient with fl-thalassemia major undergoing intravenous DFO infusion (100 mg/kg/day) for a period of 5 days.

ther reduce the possibility of iron removal from FO, analysis conditions have been adjusted such that FO elutes close to the void volume. This has the effect of reducing the contact time between FO and CP22. A simple test to confirm that no iron is removed from FO under the assay conditions was performed by injecting a solution of FO onto the column. If iron was removed, a peak corresponding to that of (CP22),-Fe (retention time = 3.12 min) would have been observed. No such peak was detected, although the peak corresponding to FO (which elutes close to the void volume) is clearly visible (Fig. 2D). A standard curve was generated by injecting known amounts of iron-NTA onto the HPLC column. A linear increase in absorbance with correlation coefficient values > 0.99 with respect to micromolar iron concentration was observed when either peak height or peak area was monitored (Fig. 3). The above behaviour equally applies for millimolar concentrations of iron. This assay which has been developed specifically for the quantification of NTBI is accurate, highly reproducible, and selective for iron. In addition, it is simple, fast, and easily automated permitting the analysis of hundreds of samples per day. With minor modifications, this method is equally applicable for measuring low molecular weight iron in other biological fluids. This assay is currently being used to assessthe progress of chelation

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