Analytical Biochemistry 394 (2009) 186–191

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Analytical Biochemistry

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Fluorescence assay of non-transferrin-bound iron in thalassemic serausing bacterial siderophore

Manisha Sharma a, Renu Saxena b, Nivedita K. Gohil a,*

a Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi 110 016, Indiab Department of Hematology, All India Institute of Medical Sciences, New Delhi 110 029, India

a r t i c l e i n f o

Article history:Received 9 March 2009Available online 24 July 2009

Keywords:TransferrinNTBIFluorescenceAzotobactinThalassemia

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.07.028

* Corresponding author. Fax: +91 11 2658 2037.E-mail addresses: nkar@netearth.iitd.ernet.in, nkar

1 Abbreviations used: NTBI, non-transferrin-boundpercentage transferrin saturation; SF, serum ferritin; Hchromatography; Fl-aTf, fluorescence-labeled apotranlabeled desferoxamine; EDTA, ethylenediaminetetraacacid disodium salt; aTf, apotransferrin; BPT, bathopheniron binding capacity; TIBC, total iron binding capacitmean; SD, standard deviation; SAE, standard analytransferrin.

a b s t r a c t

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence,a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and val-idation of treatment regimens for iron overload, is important. An assay based on iron chelation by ahigh-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of nativeapotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finallythe addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe3+ to azotob-actin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 seraranged from 0.07 to 3.24 lM (0.375 ± 0.028 lM [means ± SEM]). It correlated well with serum iron andpercentage transferrin saturation but not with serum ferritin. Pearson’s correlation coefficients werefound to be 0.6074 (P < 0.0001) and 0.6102 (P < 0.0001) for percentage transferrin saturation and totalserum iron, respectively. The low values are due to the patients being under regular chelation therapyeven prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a muchlower detection limit than the available methods.

� 2009 Elsevier Inc. All rights reserved.

Thalassemia is a type of hemoglobinopathy resulting from under-expression of the polypeptide chains in hemoglobin. It is character-ized by reduced or sometimes no synthesis of a- or b-globin chain.Consequently, the amount of hemoglobin produced is less, althoughthe amount produced is normal. Thalassemias are prevalent in manyparts of the world, and in India the incidence of both a- and b-thal-assemia is particularly high in the eastern region. The only courseof treatment for the severe anemia in thalassemia patients is re-peated blood transfusion. Although transfusions are life-saving insuch patients, they ultimately cause iron overload. Other diseasesassociated with transfusional iron overload are sickle cell anemia,aplastic anemia, myelodysplastic syndromes, and improper dietaryabsorption of iron as exemplified by hereditary hemochromatosis.

The overload appears as excess iron in the serum and is collec-tively known as non-transferrin-bound iron (NTBI).1 This is in

ll rights reserved.

m@yahoo.com (N.K. Gohil).iron; SI, serum iron; %TS,

PLC, high-performance liquidsferrin; Fl-DFO, fluorescein-

etic acid; NTA, nitrilotriaceticanthroline; UIBC, unsaturatedy; SEM, standard error of thetical error; hTf, iron-bound

excess of the iron binding capacity of transferrin, resulting in itsbinding to various proteins and other putative ligands in the circula-tion. NTBI levels vary between 1 and 10 lM in overload patients [1].NTBI is potentially toxic because it generates free radical formation.Persistent levels of plasma NTBI lead to deposition of excess iron intissues, particularly in the liver, endocrine glands, and heart, leadingto various pathophysiological conditions. Iron overload is diagnosedindirectly by estimating total serum iron (SI), percentage transferrinsaturation (%TS), and transferrin iron binding capacity by physico-chemical methods, serum ferritin (SF) levels by immunoassay and li-ver biopsy. Although these methods are quite effective in detectingsevere iron overload, they do not accurately reflect low levels of ironoverload, and liver biopsy is not desirable because it is an invasivemethod. Furthermore, studies have also shown that in hemochroma-tosis patients, NTBI is present in spite of incomplete transferrin sat-uration [2]. Therefore, these classical parameters might not providean accurate picture of the iron status of patients. For this reason, it isimportant to monitor and accurately quantify the NTBI fraction. An-other application lies in monitoring the efficacy of iron chelationtherapy in established cases of iron overload.

Currently, there is no generally accepted routine clinical assay forthe accurate quantification of NTBI, particularly in India. At the re-search level, large variations have been observed for a number ofmethods already described for quantification of NTBI. The test

Fluorescence assay of non-transferrin-bound iron / M. Sharma et al. / Anal. Biochem. 394 (2009) 186–191 187

principles of these methods can be roughly divided into two groups.The first group of methods mobilizes NTBI by a shuttle molecule,such as a low-affinity iron chelator, followed by separation of theserum proteins from the chelated iron. The chelated fraction is thenanalyzed by high-performance liquid chromatography (HPLC) [3] oratomic absorption and inductive conductometric plasma mass spec-trometry [4]. Although these detection methods have high reliabili-ties, they are difficult to set up in nonspecialized laboratories. Thesecond group of methods mobilizes and detects NTBI in the samereaction mixture without separation of the serum proteins from che-lated iron. These methods employ mainly iron-sensitive fluores-cence probes, such as fluorescence-labeled apotransferrin (Fl-aTf)[5] and fluorescein-labeled desferoxamine (Fl-DFO) [6], to quantifyNTBI in a 96-well plate setup. A limitation of these methods is theirtendency to be affected by serum color and turbidity; therefore, theymight not be discerning, particularly at low NTBI levels.

In view of the above scenario, there is a widely recognized needfor a highly sensitive and accurate clinical assay for estimation ofNTBI devoid of said limitations that is cost-effective, providinghigh-throughput efficiency and having broad clinical applicationin both the diagnosis and validation of treatment regimens for ironoverload conditions. The main focus of this study was on the appli-cation of naturally occurring fluorescent probes as analytical toolsfor assessing the free iron levels in biological fluids. Siderophoresexpressed by certain bacteria (Pseudomonas and Azotobacter gen-era) under iron-deprived conditions fall into this category and havehigh affinity (K = 1020–1052) for iron [7]. In this study, we demon-strate the feasibility of accurate determination of serum NTBI usingthe siderophore azotobactin from Azotobacter vinelandii and de-scribe the detailed protocol for the same.

Materials and methods

Experimental

Reagents and solvents commercially purchased were of analyti-cal grade and used without further purification. They included ironstandard solution (E. Merck, Germany), cobalt chloride hexahydrate,ethylenediaminetetraacetic acid (EDTA, Merck, India), nitrilotriace-tic acid disodium salt (NTA, Acros Organics, USA), bathophenanthr-oline disulfonic acid disodium salt (Loba-Chemie, India), Mopsbuffer [3-(N-morpholinopropanesulfonicacid)], ammonium acetate(SRL, India), thioglycolic acid (Sigma, USA), a,a0-bipyridyl (BDH Lab-oratory, India), potassium chloride, disodium hydrogen phosphateanhydrous, potassium dihydrogen phosphate anhydrous (S.D. FineChemicals, India), and serum ferritin immunometric enzyme immu-noassay (ORGENTEC Diagnostika, Germany).

Production of azotobactin, isolation, and purification

The peptidic siderophore was isolated from suspension culturesof two strains of A. vinelandii (wild strain NCIM-2821 [ATCC 12837,National Chemical Laboratory, India] and mutant strain F-196 pro-vided by William Page [University of Alberta, Canada]) according toa previously described procedure [8] with slight modifications. Thepurity of each fraction was established using HPLC, with the spec-trum at 380 nm consisting of a single narrow peak. Furthermore, itwas shown to fluoresce maximally at 490 nm when excited at380 nm [8].

Assay procedure for estimation of NTBI in human serum

PatientsThe patient population consisted of 42 males and 21 females

ranging between 2 and 25 years of age and suffering from b-thalas-

semia major. Most of the patients were receiving regular bloodtransfusions and were under chelation therapy. The protocol forthe study included phlebotomy only and was approved by the eth-ics committee of the All India Institute of Medical Sciences, India.Informed consent was obtained from all participating patients ortheir parents. Serum was separated within 1 h of collection toavoid the possible release of iron from hemolysis of erythrocytesand was stored at –20 �C until further analysis.

Test protocolThe principle of the assay is based on mobilizing NTBI, which is

bound to other putative ligands in the serum by a mild chelator [9].Prior to mobilization, the endogenous apotransferrin iron bindingsites (which are Fe3+ free) must be blocked or saturated so thatthe mobilized NTBI is not scavenged by apotransferrin (aTf), whichwould otherwise lead to an underestimation of NTBI. This wasdone with a trihydrate salt solution of cobalt(III). The next step in-volves the addition of NTA, a chelator with moderate affinity forFe3+, to scavenge iron bound weakly to ligands in serum other thantransferrin. Following this is a separation step to remove all serumproteins, thereby retaining the filtrate with its NTA-bound Fe3+

only. Finally, an excess concentration of the probe molecule azot-obactin is added and, having higher affinity for iron than the shut-tle NTA, extracts all Fe3+ from it. Azotobactin interacts with themobilized iron, and the quenching of its fluorescence is measuredas a function of iron (Fe3+) concentration.

A trihydrate salt solution (80 mM stock) of cobalt(III), preparedfrom CoCl2 according to a previously described method [10], wasused as the blocking agent. A stock solution of NTA (800 mM) inMilli-Q water at pH 7.0 was used as the mobilizing agent. To deter-mine the optimum concentration of NTA, different aliquots of thestock were used to achieve the final concentrations of 10, 50, 80,100, 120, 140, and 160 mM NTA and checked with standard ironadded to normal serum to give final concentrations of 0.2 and0.4 lM Fe3+. Azotobactin solution (0.85 lM) prepared in acetatebuffer at pH 4.4 was used as the probe.

AssayIn an Eppendorf tube, serum and cobalt(III) solution were mixed

in the ratio of 4.5:1. The contents were vortexed, mixed well, andincubated for 30 min at 37 �C. Further NTA solution was added tothe serum mixture to obtain the final concentration of 80 mM. Thevolume was made up to 1.0 ml by sodium phosphate buffer (pH7.4), mixed well, and allowed to stand for 30 min at room tempera-ture. The serum mixture was then ultrafiltered (Centrikon-10, Milli-pore, USA) at 8000 rpm for 20 min. Next, the ultrafiltrate andazotobactin solution were mixed in the ratio of 2.9:1 in a disposablefluorescent cuvette and allowed equilibration time of 10 min, afterwhich the fluorescence emission intensity was measured.

EquipmentFluorescence spectra were recorded on an LS 50B luminescence

spectrometer (PerkinElmer, UK). The data were processed onlineusing FL WinLab software. The excitation/emission wavelengthused was 380/490 nm, with the excitation and emission slit widthsoptimized to 2.5 and 5.0 nm, respectively.

Construction of iron calibration curveThe calibration curve for the estimation of NTBI or free iron was

prepared in human serum obtained from normal subjects. Aliquotsof normal serum were prepared with increasing concentrations ofiron by the addition of standard iron solution. Furthermore, theserum was processed in the same manner as described in the assayprocedure with cobalt(III) as the blocking agent and 80 mM NTA asthe mobilizing agent. The concentration range used for the gener-ation of the calibration curve was 0–0.6 lM. A plot of the ratio of

188 Fluorescence assay of non-transferrin-bound iron / M. Sharma et al. / Anal. Biochem. 394 (2009) 186–191

fluorescence versus input iron concentration was generated. Thebest fit was obtained by nonlinear regression analysis using theexponential association model represented as y = a[b � exp(�cx)],where a, b, and c are constants.

NTBI measurement by colorimetric methodNTBI was also measured in 41 samples by a standard colorimet-

ric method for iron estimation that is based on bathophenanthro-line (BPT). To the ultrafiltrate obtained as before was addedMops buffer (5 mM, pH 7.4) in a 1:1 ratio to make a total volumeof 800 ll. Then 100 ll each of thioglycolic acid (120 mM) andBPT (60 mM) was added to this, mixed, and allowed to stand for30 min. After incubation, the absorbance was measured at 537 nm.

Clinical parametersSerum samples were also tested for classical parameters such as

SI, unsaturated iron binding capacity (UIBC), and SF. SI and UIBCwere estimated by colorimetric assays using a,a0-bipyridyl as thechromogenic agent [11]. Total iron binding capacity (TIBC) and%TSwere calculated by the following formulas:

TIBC ¼ SIþ UIBC%TS ¼ SI=TIBC� 100:

SF levels were estimated by an enzyme immunoassay.

Statistical analysesStatistical analysis was performed with GraphPad Prism 5 soft-

ware. Data are presented as means ± standard errors of the mean(SEMs) unless stated otherwise, and P < 0.05 was considered assignificant.

Results

The fluorescence excitation/emission wavelength of azotobactinwas found to be 380/490 nm (Fig. 1A). On binding with iron, theintensity of fluorescence is quenched in a concentration-depen-dent manner (Fig. 1B). Based on this observation, the calibrationcurve was drawn in serum derived from healthy volunteers(Fig. 2). Serum was processed as in the test protocol and, therefore,the final sample did not contain any proteins. NTA at a concentra-tion of 80 mM was proposed previously [9] to remove all forms ofNTBI. It may also mobilize a small fraction of the iron bound totransferrin. However, this will depend on the duration of incuba-tion. Fig. 3 shows the effect of different NTA concentrations on iron

Fig. 1. (A) Fluorescence spectra of azotobactin showing excitation at 380 nm (left spectr4.4). (B) Quenching of emission on binding with a range of micromolar concentrations ofAZ, azotobactin.

extraction in the case of two known added iron levels to normalserum. It is seen that there is a steady increase in iron mobilizationin the case of both 0.2 and 0.4 lM up to 80 mM NTA. Beyond80 mM there is essentially no further increase. The values of NTBImeasured in clinical samples were extrapolated from the calibra-tion curve. The NTBI levels obtained with the current method ran-ged from 0.07 to 3.24 lM (0.375 ± 0.028 lM, n = 63), whereasthose measured by the BPT method ranged from 0 to 1.54 lM(0.2068 ± 0.0350 lM, n = 41). The summary of the measured clini-cal parameters is given in Table 1. With the exception of SF, NTBImeasured by this method correlates well with all classical param-eters (Figs. 4 and 5). Pearson’s correlation coefficients were foundto be 0.6074 (P < 0.0001) and 0.6102 (P < 0.0001) for %TS and SI,respectively. Of the 63 cases, 7 had overt symptoms of cardiomy-opathy, impaired growth, and hepatic cirrhosis. Interestingly, inthese patients, transferrin was not fully saturated (25.06 ± 2.76%),yet NTBI was higher (0.677 ± 0.108 lM).

The accuracy and precision of this developed analytical assaywas confirmed with solutions of different concentrations of iro-n(III) analyzed in triplicate. All of the samples were processed inthe same manner as described in the assay procedure with cobal-t(III) as the blocking agent and 80 mM NTA as the mobilizing agent.The results are summarized in Table 2. The means ± standard devi-ations (SDs) were considered as satisfactory for the range of quan-tities of iron(III) examined. The inter- and intraday precision valuesof the assay were analyzed on a random selection of 10 thalassemicpatients on three separate days and at three separate times on thesame day. The serum samples were frozen during the intervals. Theintra- and interday precision was defined as the SD, and accuracywas determined by calculating the standard analytical error(SAE). The SDs of individual NTBI measurements varied from 0.01to 0.17 in the case of interday measurements, whereas they variedfrom 0 to 0.5 in the case of intraday measurements. The accuracyvalues calculated as the SAEs of inter- and intraday measurementswere within the range of 0.5–10%.

Discussion

The high sensitivity of fluorescent techniques coupled with theselectivity of receptor–ligand interactions has become the mostwidely accepted analytical tool as compared with other spectro-metric methods. Fluorescent molecules of various types have beenused in the past to sense intracellular iron as well as extracellulariron present as NTBI [12,13]. Some of these methods essentially

um) and emission at 490 nm (right spectrum) with 0.05 mol L�1 acetate buffer (pHFe3+: 0.06, 0.133, 0.2, 0.266, 0.33, 0.40, 0.466, 0.533, and 0.6 lM. a.u., arbitrary units;

Fig. 2. Calibration curve derived by the addition of a series of concentrations ofstandard iron to normal serum. F0/F is the ratio of intensity of native azotobactin tointensity of the azotobactin–iron complex.

Fig. 3. Effect of different concentrations of NTA on iron chelation from nonspecificligands in serum. The effect was studied in aliquots of normal serum containing 0.2and 0.4 lM standard iron.

Fig. 4. Graph illustrating a linear correlation between NTBI levels and SI levels inclinical samples.

Fig. 5. Graph illustrating a linear correlation between NTBI levels and %TS in clinicalsamples.

Fluorescence assay of non-transferrin-bound iron / M. Sharma et al. / Anal. Biochem. 394 (2009) 186–191 189

require that the iron-capturing molecule be labeled by a fluoro-phore for detection of iron binding. The current assay employs anintrinsically fluorescent bacterial siderophore, azotobactin, forsensing NTBI. The novelty lies in its being an iron chelator itselfalong with the presence of the fluorescent chromophore as partof the molecule. Azotobactin is one pyoverdine-type siderophoreamong others secreted by Azotobacter species of bacteria. It is a yel-low–green fluorescent O-dihydroxyquinoline peptide [14,15]. Thehexadentate ligand binds to iron (Fe3+) firmly, giving very stableoctahedral complex that is nonfluorescent in nature [8]. BecauseFe3+ quenches fluorescence of azotobactin on complexation, theconcentration of iron can be estimated by measuring the decreasein the fluorescent signal. This underlying characteristic phenome-non of quenching of fluorescence was used in the proposed assayfor estimating NTBI in human serum.

Careful selection of mobilizing and blocking agents were madeto ensure that these molecules did not alter or influence the forma-

Table 1Means ± SEMs of serum parameters SI, UIBC, TIBC, %TS, and SF in 63 patients.

SI (lg/dl) UIBC (lg/dl) TIBC (lg/dl) %TS

N 63 63 63 63Mean 88.14 250.0 339.8 26.19SEM 5.03 5.16 1.22 1.56

tion of azotobactin–Fe3+ complex in any way and further did notalter the fluorescent characteristics of azotobactin. Various mobi-lizing agents, such as sodium oxalate, NTA, and EDTA, have beendescribed before. Fluorescence emission of azotobactin in the pres-ence of sodium oxalate, NTA, and EDTA showed that iron–NTAcomplex transfers iron rapidly to azotobactin, which is markedby a decrease in fluorescence emission intensity and increasedquenching of the latter, as observed in Fig. 6. Iron complexed to so-dium oxalate and EDTA, however, did not show any quenching fol-lowing incubation with known concentrations of azotobactin. Inview of recent reports [16], we also examined the effect of differentconcentrations of the low-affinity chelator NTA on iron mobiliza-tion from serum. NTA was selected on the basis that it has suffi-cient binding affinity to remove all iron nonspecifically bound toserum proteins and other ligands. At the same time, it would bereasonable to assume that it does not remove significant amountsof iron bound to transferrin, particularly within the short incuba-tion time of 30 min. In time course experiments done previously

SF (ng/ml) NTBI (lM): azotobactin assay NTBI (lM): BPT assay

63 63 412228 0.375 0.207

284.5 0.028 0.035

Table 2Nominal and determined concentrations of samples.

Sample Nominal concentration (lM) Determined concentration (lM)a

1 0.2 0.21 ± 0.012 0.6 0.56 ± 0.043 1.0 1.04 ± 0.13

Note. The reproducibility of the method was tested in triplicate in aliquots of nor-mal serum containing fixed amounts (0.2, 0.6, and 1.0 lM) of standard iron solu-tion. This serum was processed as in Materials and methods and assayed with0.85 lM azotobactin solution.

a Means ± SDs, n = 3.

Fig. 7. Graph illustrating correlation between NTBI levels measured by theazotobactin assay and BPT method in clinical samples.

Fig. 6. Effect of different chelators—NTA, sodium oxalate (SO), and EDTA—on ironbinding to azotobactin and its fluorescence quenching.

190 Fluorescence assay of non-transferrin-bound iron / M. Sharma et al. / Anal. Biochem. 394 (2009) 186–191

[9], it has been demonstrated clearly that incubation of holotrans-ferrin (iron-bound transferrin [hTf]) with 80 mM NTA for up to72 h leads only 10% of hTf to lose iron. Our incubation period isonly 30 min, which is likely to extract a negligible amount of ironfrom transferrin. For the same reason, a flat profile is observed be-tween 80 and 160 mM NTA in Fig. 3. It was concluded from the re-sults that an 80-mM concentration was the upper limit at which allnonspecifically bound iron was extracted in the serum under thegiven experimental conditions. Any further increase in the concen-tration of NTA did not lead to an increase in extracted iron. Further,it donates easily the bound iron to fluorescent probe moleculeazotobactin. This can be inferred from the values measured inthe case of the calibration curve where normal serum was spikedwith known concentrations of standard iron. The measured valueswere within 5%.

This assay incorporates a step for separation of the proteins, asin Gosriwatana and coworkers [9], for two specific reasons. Thefirst is to eliminate the presence of hTf in the measuring solutionbecause azotobactin does extract Fe3+ from transferrin (our obser-vation not reported here). The second reason is that the absence ofall proteins in the measuring solution is important for fluorimetricassays because it eliminates the background noise contributed byprotein fluorescence, resulting in higher sensitivity. This is theadvantage of the developed method as compared with the one-step fluorescent assay developed by Breuer and Cabantchik [5].The issue of variation in color and turbidity of serum sampleswas addressed in their method by taking the ratio of change in sig-nal obtained due to binding of iron with fluorescent apotransferrin(Fl-aTf) and other unknown factors in the sample (presumably pro-teins) to the change in signal caused only by the unknown factorsin the presence of excess aTf. However, under these conditions, thepossibility of competitive binding of iron between added apotrans-

ferrin and Fl-aTf cannot be ruled out completely. Another source ofambiguity arises from the use of gallium(III) as the blocking agent.It is shown to enhance the quenching of Fl-aTf by concentrations ofiron in the range of 0–12.5 lM if added to Fl-aTf either before ortogether with iron. Because both reagents A and B in the assay con-tain gallium as part of the kit, it is available to Fl-aTf before theaddition of the serum sample containing NTBI (iron) and, therefore,would contribute to the enhancement of fluorescence quenching inthe test sample. Following on similar lines, we could not use gal-lium(III) as blocking agent for native apotransferrin because theazotobactin–iron interaction was influenced in the presence of gal-lium (results not shown).

NTBI is reported to be present at concentrations up to 10 lM[1]. The values reported in this cohort of patients are fairly low.It is unlikely that there would be any unfilterable component[16] in our samples given that NTA was used to mobilize ironand the molecular mass cutoff of the ultrafiltration units usedwas 10 kDa. The low values can be attributed to the fact that allof the patients were under regular chelation therapy even priorto sampling, unlike in other studies where chelation typically isstopped 24–48 h before the sample is withdrawn [2,17]. The pa-tients were administered a daily dose of three to nine tablets ofKelfer (deferiprone). Concomitantly, the %TS values also reflectchelation. In view of this, it is encouraging to note that the probeazotobactin is sensitive to such low levels of NTBI, indicating itsexcellent suitability for monitoring low to moderate iron overloadconditions. Only one patient who had not been put on chelationwas available; the corresponding NTBI and%TS values were3.24 lM and approximately 80%, respectively. These data are notincluded in Table 1.

Furthermore, NTBI measured by this method showed a good po-sitive correlation with SI and %TS. No correlation with SF was ob-served, probably because the latter is not specific for thalassemiaand is known to be elevated in any inflammatory condition.

Of the 63 patients in the sample population, an interestingobservation was made in the cases of 7 patients who also showedovert symptoms of cardiomyopathy, hepatic cirrhosis, and im-paired growth. They also were on regular chelation therapy with25% transferrin saturation. Yet the NTBI values in these specificcases were higher than the rest. This indicates that in the total poolof NTBI, some fractions were probably less/slowly chelatable bythe drug. This observation is supported by the suggestion by Evansand coworkers [17] that different forms of NTBI in the heteroge-neous pool vary with the disease state. It also indicates that %TSby itself might not be a precise index of iron overload. In fact,

Fluorescence assay of non-transferrin-bound iron / M. Sharma et al. / Anal. Biochem. 394 (2009) 186–191 191

the correlation data improve to 0.726 and 0.723 (P < 0.0001) for%TS and SI, respectively, if these 7 cases are treated separately.

The method was compared with the BPT colorimetric method ofiron estimation. It was observed that the NTBI values measured bythe colorimetric method tend to be lower than those obtained bythe developed assay (P < 0.0001 in a paired t test, n = 41) eventhough the same protocol was used for NTBI extraction. The quan-tification of NTBI by BPT is totally dependent on the conversionrate of ferric ions to ferrous ions, which then forms the chromo-genic complex with BPT that is measured. The low values of NTBIas estimated by the BPT method may be due to incomplete conver-sion of ferric ions to ferrous ions causing underestimation of NTBI.Moreover, both cobalt and the iron complexed metabolites of che-lators present in serum of patients undergoing chelation therapymay be interfering with the colorimetric reagent used in this test.The Pearson’s correlation coefficient between the two methodswas found to be 0.89 (P < 0.0001), as shown in Fig. 7.

In conclusion, our aim in this study was to develop a new assay forNTBI that includes all chemically and functionally heterogeneousforms. In addition, we have demonstrated that this is useful inassessing the levels of NTBI precisely and accurately not only in se-vere iron overload cases but also in cases where low to moderate lev-els of NTBI exist when the patient is put on chelation therapy. Theprincipal advantage of the proposed method over existing methodslies in its simplicity and sensitivity, permitting the analysis of largenumbers of patient samples. Validation of the method in other ironoverload conditions will be conducted in the near future.

Acknowledgments

Financial assistance from the Indian Institute of Technology (IIT,New Delhi) is gratefully acknowledged. Manisha Sharma is sup-ported by a student fellowship from IIT. We are grateful to WilliamPage for providing us with the mutant strain of A. vinelandii.

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