Effects of in vitro glycation on [Fe.sup.3+] binding and [Fe.sup.3+] isoforms of transferrin.
In addition to its role in regulating the iron fluxes between the sites of absorption, storage, and utilization, Tf has also been attributed a very important antioxidant function in plasma. Free [Fe.sup.2+] is very reactive and capable of producing free radicals that cause oxidative damage to biomolecules (5). Typically, no free [Fe.sup.2+] is present because all iron is bound to Tf in a redox-inactive [Fe.sup.3+] form. The exact ways in which modifications or abnormalities of Tf affect its [Fe.sup.3+]-binding and antioxidant capacity are not yet fully elucidated.
In diabetes mellitus, protein structure and function are significantly affected by glycation. The aldehyde group of glucose reacts nonenzymatically with amino groups of proteins to form Schiff's bases, which undergo Amadori rearrangements, forming fructosamines. These modified proteins degrade slowly and irreversibly to advanced glycated end products (6). Because diabetes is also associated with both an increase in oxidative stress (7, 8) and disturbances in iron metabolism (9-11), it is of great interest to investigate the hypothesis that glycation of Tf can contribute to oxidative stress by impairment of its antioxidant function. In a recent study we showed that in vitro glycation of Tf decreases its [Fe.sup.3+]-binding capacity and makes it less effective in protecting against in vitro lipid peroxidation (12). Therefore, in the present study we aimed to gain more insight into the mechanisms underlying the effects of glycation on the [Fe.sup.3+]-binding capacity of Tf. For this purpose, we investigated the distribution of [Fe.sup.3+] between the two binding sites by studying the IEF patterns of in vitro-glycated Tf.
Materials and Methods
IN VITRO GLYCATION OF Tf
Human apoTf (purity [greater than or equal to]97%, iron free; cat. no. T2036; Sigma-Aldrich) at a concentration of 5 g/L was dissolved in sodium phosphate buffer (0.1 mol/L, pH 7.4) containing different concentrations of D-glucose (0, 5.6, 13.9, 22.2, 33.3, or 1000 mmol/L) and incubated for 14 days at 37[degrees]C under sterile conditions. Thereafter, the remaining free glucose was removed by passing the reaction mixtures through a Sephadex G-25 column (PD-10; Amersham Bioscience) equilibrated with sodium phosphate buffer. The protein concentration in each of the collected fractions was determined by the Bradford method (13) with weighed solutions of apoTf as calibrators. Aliquots of 120 [micro]L (3 g/L) were stored at -70[degrees]C until further use. Tf integrity after storage was verified nephelometrically (BN II Nephelometer; Dade Behring) with specific antibodies against Tf (CSAX 15) and calibrated against a commercial standard (CRM 470, IFCC-validated).
We determined the extent of glycation by measuring the fructosamine concentration with the nitroblue tetrazolium colorimetric assay (assay A11A00350, Cobas Mira; ABX Diagnostics). This method is based on the reducing ability of fructosamines in alkaline solution. At 37[degrees]C the sample is added to carbonate buffer (pH 10.35) containing nitroblue tetrazolium, which is subsequently reduced to formazan. This causes an increase in the absorbance at 550 nm that is measured spectrophotometrically between 10 and 15 min.
[Fe.sup.3+] BINDING AND IEF OF [Fe.sup.3+]-Tf ISOFORMS
To identify the conditions for efficient [Fe.sup.3+] binding, we incubated 100 [micro]L of apoTf in sodium phosphate buffer, pH 7.4 (final concentration, 2.5 g/L), with 10 [micro]L of sodium bicarbonate (final concentration, 30 mmol/L) and 10 [micro]L of different concentrations of freshly prepared Fe[Cl.sub.3], iron citrate, or Fe:nitrilotriacetic acid [Fe:NTA, molar ratio 1:7, prepared by adding equal volumes of 10 mmol/L ferrous ammonium sulfate and 70 mmol/L NTA (adjusted to pH 7.0) according to the specifications of Breuer and Cabantchik (14)]. The pH of the reaction mixture was stable at 7.4. Final [Fe.sup.3+] concentrations of 0, 20, 40, 60, 150, 300, 600, and 1000 [micro]mol/L theoretically achieved Tf saturations of 0%, 32%, 64%, 96%, 240%, 480%, 960%, and 1600% as calculated by the formula:
Tf saturation = iron concentration/TIBC
Where the total iron-binding capacity (TIBC) = 25 x the Tf concentration, in g/L (15).
The time course of the formation of the Tf-Fe complexes was monitored spectrophotometrically either in cuvettes at 470 nm (UV2 Series; Unicam) or in 96-well plates at 450 nm ([EL.sub.x]808 Ultra Microplate Reader; BioTek Instruments Inc.).
To avoid interference in the IEF band pattern of [Fe.sup.3+]-Tf isoforms, we removed sialic acid residues enzymatically by adding neuraminidase (NA; Clostridium perfringens; Beckman Coulter Inc.). Varying the incubation time revealed that the most efficient removal occurred after overnight incubation of apoTf with a concentration of 0.1 U NA/mg of Tf. This was illustrated by shifts in the bands toward the cathode (higher pI) with increasing duration of incubation (Fig. 1).
IEF of the different Tf solutions was performed on the PhastSystem[TM] with PhastGel IEF pH 5-8 (Pharmacia LKB). The PhastSystem Separation Technique File No. 100 (from the User's Manual) was used with slight modification of the standard program according to van Noort et al. (16):
Sample applicator down at 1.2 0 V x h
Sample applicator up at 1.3 0 V x h
Extra alarm to sound at 1.1450 V x h
Sep 1.1: 2000 V, 2.0 mA, 3.5 W at 15[degrees]C for 585 V x h
Sep 1.2: 200 V, 2.0 mA, 3.5 W at 15[degrees]C for 15 V x h
Sep 1.3: 2000 V, 5.0 mA, 3.5 W at 15[degrees]C for 450 V x h
Eight samples were applied per gel with an applicator that was placed at the cathodic end of the gel between the extra alarm and Sep 1.2. Immediately after focusing, the gels were fixed for 10 min in 100 g/L trichloroacetic acid and stained overnight by incubation with EZBlue[TM] Gel Staining Reagent (cat. no. G1041; Sigma-Aldrich) and washed in distilled water. The isoforms were identified by measuring the distance of each band from the cathode and calculating the pI from a calibration line that was generated with pI markers (calibration set pH 5.20 to 7.35; Amersham Pharmacia Biotech UK Limited). On the basis of the knowledge that binding of each [Fe.sup.3+] causes a decrease in pH units (~0.2) double that caused by each sialic acid residue (~0.1) and according to the diagrammatic representation of de Jong and van Eijk (17), we assigned each pI value a isoform identification as shown in Fig. 2.
[FIGURE 1 OMITTED]
Digital images of the Phastgels were obtained by scanning with a HP Scanjet 4P, and the relative distribution of the different isoform bands was assessed with use of UN-SCAN-IT gel[TM] digitizing software for Windows (Ver. 5.1; Silk Scientific Inc.). The area enclosed by each peak was given as total pixel intensity and expressed as percentage of the sum of all peaks. This method demonstrated the acceptable reproducibility and stability of the frozen Tf samples. Quantification of the isoform bands was linear at Tf concentrations of 0.187-1 g/L (16).
[FIGURE 2 OMITTED]
Results are expressed as the mean (SD). The statistical significance of the differences was evaluated by paired and unpaired Student t-tests for two-group comparisons, one-way AMOVA, and Pearson correlation coefficients to compare the different glycation conditions (Excel software). Two-tailed P values <0.05 were considered significant.
Preliminary experiments were conducted to find the optimum conditions of [Fe.sup.3+] binding before we separated the [Fe.sup.3+]-Tf isoforms by IEF.
EFFECT OF THE TYPE AND CONCENTRATION OF [Fe.sup.3+] COMPOUND ON [Fe.sup.3+] BINDING
In a first series of experiments, we investigated the optimum conditions for [Fe.sup.3+] binding. Various [Fe.sup.3+] compounds at different concentrations were incubated with fresh apoTf up to 18 h, and formation of the iron-Tf complex was monitored at 470 run. Binding was highest in the case of Fe:NTA. For example, after 18 h, the absorbance was 0.041, 0.055, and 0.063 arbitrary absorbance units for Fe[Cl.sub.3], iron citrate, and Fe:NTA, respectively, at 32% theoretical [Fe.sup.3+] saturation. At 96% theoretical [Fe.sup.3+] saturation, these values were 0.117, 0.103, and 0.173 absorbance units, respectively.
After overnight incubation of apoTf with these different [Fe.sup.3+] compounds, the samples were subjected to IEF to separate the different [Fe.sup.3+]-Tf isoforms. As illustrated in Fig. 3, there was a shift of the bands toward the anode with increasing [Fe.sup.3+] concentrations. This shift from apoTf (cathodal position) toward Tf with one or two [Fe.sup.3+] ions bound (more anodal position) was observed for the three [Fe.sup.3+] compounds but was more pronounced for Fe:NTA. Fe2-Tf represented 6 (6)%, 30 (13)%, and 66 (27)% at 32%, 64%, and 96% theoretical [Fe.sup.3+] saturation. The isoform proportion in the N-band was not different from that in the C-band: respectively, 21.9 (4.0)% and 22.2 (5.8)% at 32% theoretical [Fe.sup.3+] saturation, 20.8 (2.8)% and 26.3 (7.6)% at 64% theoretical [Fe.sup.3+] saturation, and 12.1 (9.3)% and 17.2 (13.3)% at 96% theoretical [Fe.sup.3+] saturation (not significantly different in the paired comparison; n = 5). In subsequent experiments, the effect of glycation on [Fe.sup.3+] isoforms of Tf was investigated after overnight incubation of nonglycated or glycated Tf with both NA and Fe:NTA.
EFFECTS OF GLYCATION
Incubation of human apoTf [half-life =7 days (18)] for 14 days with different concentrations of glucose led to a concentration-dependent increase in the fructosamine content (13.0, 15.5, 22.5, 47.0, and 102.5 [micro]mol/L after incubation with 0, 5.6, 13.9, 22.2, and 33.3 mmol/L glucose, respectively). These glycated Tf solutions were incubated with three different concentrations of Fe:NTA to obtain theoretical [Fe.sup.3+] saturations of 32%, 64%, and 96%.
[FIGURE 3 OMITTED]
Time course of [Fe.sup.3+] binding. We monitored [Fe.sup.3+] binding spectrophotometrically by measuring the increases in absorbance at 450 run after subtraction of the blank reaction with no iron added (Fig. 4). The mean (SE) increase in absorbance after 90 min was 0.038 (0.005), 0.051 (0.004), and 0.066 (0.005) absorbance units for the various Tf solutions (n = 42) at, respectively, 32%, 64%, and 96% theoretical [Fe.sup.3+] saturation. There was no further significant increase in absorbance during the overnight incubation. We observed no difference between fresh Tf and Tf preincubated for 14 days in the absence of glucose. In contrast, glycation of Tf tended to decrease [Fe.sup.3+] binding, but this difference reached statistical significance only for the Tf glycated with 1 mol/L glucose (P <0.05 compared with Tf preincubated in the absence of glucose).
[Fe.sup.3+]-Tf isoforms. After glycation and additional incubation in the presence of Fe:NTA, the different [Fe.sup.3+]-Tf isoforms were separated by IEF. A representative example is shown in Fig. 5. The percentage distribution of the various [Fe.sup.3+]-Tf isoforms was not significantly different between the fresh apoTf and apoTf incubated for 14 days without glucose. Preincubation with an extremely high glucose concentration (1 mol/L) produced a diffuse pattern with no detectable individual isoforms. The results of the quantitative analysis of the IEF patterns by densitometry are summarized in Fig. 6. Increasing concentrations of Fe:NTA also led to more [Fe.sup.3+] binding in glycated Tf as indicated by the increasing percentages of Fe2-Tf at the expense of Fe0-Tf. For example, at 32%, 64%, and 96% theoretical [Fe.sup.3+] saturation, the mean Fe2-Tf in the preincubated Tf solutions (n = 15) represented, respectively, 15 (4)%, 36 (5)%, and 76 (14)%, and Fe0-Tf represented 38 (7)%, 14 (6)%, and 0% (not detectable) of all isoforms. When we compared the Tf glycated at different glucose concentrations with the Tf preincubated without glucose, at a theoretical [Fe.sup.3+] saturation of 32%, Fe2-Tf was significantly higher after the glycations performed at 13.9, 22.2, and 33.3 mmol/L glucose [13 (3)%, 17 (3)%, and 18 (3)%, respectively, vs 12 (4)% for the nonglycated Tf; P = 0.06, 0.02, and 0.01 respectively; n = 3]. At the higher theoretical [Fe.sup.3+] saturation of 64%, the proportion of Fe2-Tf was not significantly different among the various glycation conditions. At 96% theoretical [Fe.sup.3+] saturation, in contrast, Fe2-Tf decreased linearly with increasing glycation [83 (14)%, 75 (16)%, 72 (21)%, and 68 (11)% after preincubation with 5.6,13.9, 22.2, and 33.3 mmol/L glucose, respectively; r = 0.97; P = 0.008]. In contrast to the results obtained with freshly prepared Tf, the proportions of the Fe1N-Tf and Fe1C-Tf isoforms were influenced by the 14-day preincubation depending on the [Fe.sup.3+] saturation. At 32% theoretical [Fe.sup.3+] saturation, the proportions of Fe1N-Tf and Fe1C-Tf were 23.9 (1.7)% and 23.0 (1.0)%, respectively (not significant). At 64% theoretical [Fe.sup.3+] saturation, the proportion of Fe1N-Tf was higher [27.3 (0.7)%] than the proportion of Fe1C-Tf isoform [23.1 (1.1)%; P = 0.009; n = 15]. At 96% theoretical [Fe.sup.3+] saturation, these proportions were 11.8 (3.0)% and 12.2 (4.2)% (not significant). This shift was not affected by the degree of glycation.
The analysis of Tf isoforms has led to abundant, often controversial literature. The separation of these isoforms is based on pI differences caused by genetic variation, iron load, and sialic acid content (4). Analysis of the sialo-Tf isoforms has already demonstrated its use as a diagnostic marker of congenital disorders of glycosylation (19) and heavy alcohol consumption (20, 21), both conditions that decrease the amount of sialylation and even lead to a loss of one or two N-glycans. In this study, we focused on the iron content of Tf as a source of its microheterogeneity. Urea gel electrophoresis has been successfully applied to differentiate between various [Fe.sup.3+]-Tf isoforms and is precise enough to detect differences in the ratio of occupancy of the N- and C-terminal sites (22). In the search for semiautomated and less time-consuming alternatives, one of the currently preferred methods is IEF because of its high resolving power. van Noort and van Eijk (16) described an IEF method using the PhastSystem that allowed fast separation and easy estimation of the relative proportions of Tf isoforms. However, and partly because of its high resolving power, interpretation of results obtained by IEF presents several hazards. For example, great care must be taken to avoid overlapping of the different types of isoforms. To overcome interference on the IEF profiles as a result of the presence of sialic acid residues, these were removed by preincubation with NA. Incubation under conditions described in the literature [0.06 U/mg of Tf at 37[degrees]C for 1 h (16)] did not succeed in removing all sialic acid residues from our Tf samples. Prolonging the incubation time and increasing the NA concentration led to more efficient removal, but even after overnight incubation with 0.1 U NA/mg of Tf, there was still a detectable monosialo-Tf isoform band. This incomplete hydrolysis has also been observed by other authors (16,23) and may be attributable to several causes, all related to the properties of this enzyme. One cause may be that the pH optimum for the enzyme is 5.8-6.0 in phosphate buffer. However, because of the importance of pH in [Fe.sup.3+]-loading experiments and the goal to mimic physiologic conditions, experiments with Tf and [Fe.sup.3+] loading were performed at pH 7.4. Another cause could be that iron, as well as other heavy metals and oxygen, inhibits the enzymatic activity of NA. Finally, the enzyme may lose activity on incubation (by as much as 50% after incubation at 37[degrees]C for 24 h) and thus be insufficient to achieve total hydrolysis at the minimal concentrations of sialylated Tf that remain toward the end of the overnight incubation.
[FIGURE 4 OMITTED]
[FIGURE 5 OMITTED]
The second methodologic concern when using IEF to separate [Fe.sup.3+] isoforms is the care that should be taken to perform optimum and accurate [Fe.sup.3+] binding, as investigated extensively by Hackler et al. (24). Although Tf is also capable of binding other ions, e.g., [Zn.sup.2+], [Ga.sup.3+], [Al.sup.3+], and [Cu.sup.2+] (2, 25-28), [Fe.sup.3+] is by far the most important metal that binds to Tf. However, as data in the literature show, [Fe.sup.3+] binding is very sensitive to changes in pH (29-31), and the presence of a synergistic anion, usually, but not necessarily, bicarbonate, is an absolute requirement (32, 33). Moreover, it is difficult to estimate the exact extent of [Fe.sup.3+] binding to apoTf, which differs among the various types of [Fe.sup.3+] salts (22, 34). We addressed this issue by investigating the binding of [Fe.sup.3+] to Tf by spectrophotometry and the formation of [Fe.sup.3+]-Tf isoforms by IEF. To mimic the in vivo situation as closely as possible, we incubated a mixture containing physiologic concentrations of apoTf (2.5 g/L) and sodium bicarbonate (30 mmol/L) with different [Fe.sup.3+] compounds at various concentrations and at a pH of 7.4. In these conditions, formation of the [Fe.sup.3+]-Tf complex was faster and more pronounced for the incubation with Fe:NTA than with Fe[Cl.sub.3] or iron citrate. In a solution at physiologic pH, the greater part of Fe:NTA exists in the monomeric form. In this way [Fe.sup.3+] is immediately available for uptake by Tf in a reaction lasting 10 s (35). Iron citrate, in contrast, forms polymeric iron complexes and thus releases low-molecular-weight iron species more slowly. Because the availability of low-molecular-weight iron is rate limiting, [Fe.sup.3+] binding to Tf requires several hours (36). When Fe[Cl.sub.3] was used, [Fe.sup.3+] binding was also less effective. Published observations show that only 5-25% of [Fe.sup.3+] binds when 1 equivalent of Fe[Cl.sub.3] is added to apoTf (34).
[FIGURE 6 OMITTED]
Each Tf molecule can bind a maximum of two [Fe.sup.3+] ions. Depending on the iron supply of the organism, Tf molecules are either iron-free or loaded with one or two [Fe.sup.3+] ions. In healthy controls, Tf saturation is ~30% and represents a mixture of four-ninths apoTf, four-ninths Tf with one [Fe.sup.3+] ion, and one-ninth Tf with two [Fe.sup.3+] ions bound. In our experiments, similar proportions were reached when Tf was incubated with [Fe.sup.3+] to achieve a theoretical saturation of 32%. Two monoferric isoforms of Tf are distinguished: Tf with [Fe.sup.3+] bound to the N-terminal binding site (Fe1N-Tf) or bound to the C-terminal binding site (Fe1C-To. In our experiments, the proportions of the Fe1N-Tf and Fe1C-Tf isoforms were similar in freshly prepared Tf. In Tf that was preincubated for 14 days in the presence or absence of glucose, a slightly higher proportion of the Fe1N-Tf isoform was observed at 64% theoretical [Fe.sup.3+] saturation. This shift was not affected by the degree of glycation. It is not known whether the two binding sites have different functional roles. This issue has led to abundant but often contradictory literature. The N- and C-terminal binding sites have similar structures, but they are not equivalent in terms of [Fe.sup.3+] uptake and release because they differ in their accessibility to iron chelates, binding strength, spectroscopic properties, kinetic lability, and response to changes in pH (29, 30, 37, 38). For example, it is known that the C-terminal site can hold [Fe.sup.3+] at a lower pH (39, 40). Fe1N-Tf binds the Tf receptor on cells with a much lower affinity than does Fe1C-Tf (41). Some reports have concluded that, in plasma, [Fe.sup.3+] is randomly distributed to the two sites (42-44), whereas others have suggested that one site is preferentially occupied (22, 45,46).
As expected, increasing concentrations of iron led to more [Fe.sup.3+] binding, as illustrated by the progressively higher percentages of the Fe2-Tf isoform and lower percentages of Fe0-Tf. Despite optimal [Fe.sup.3+] binding conditions, complete [Fe.sup.3+] saturation of Tf was not achieved. For example, at 96% [Fe.sup.3+] saturation, only the Fe2-Tf isoform should be present, but in our experiments we also observed monoferric isoforms, albeit in small amounts. When [Fe.sup.3+] saturation was calculated from the proportions of the isoform observed in the gels, values ranged from 91% in the apoTf incubated with no glucose to 83.9% in that preincubated with 33.3 mmol/L glucose. These values contrast with the theoretical 96% and suggest that during the experimental procedure (IEF) there was a loss of 5% of the nonglycated Tf and an additional 7% attributable to glycation. This observation suggests that release of [Fe.sup.3+] from the Fe2-Tf isoform might have occurred during IEF. Release might occur at the anodal, more acidic position of the Phastgel where Fe2-Tf is located because it is well known that release of iron from Tf occurs at pH <6.3 (47). In addition, sequestering of iron by the ampholytes during IEF might also occur. Zak and Aisen (22) observed a 25% loss in absorbance at 470 run of saturated transferrin in 2% Ampholine. Because of the observed difference between the expected saturation and the actual saturation, we have consistently used the term "theoretical saturation" when describing [Fe.sup.3+] concentrations in relation to Tf. In view of this observation, extrapolation of in vitro results of iron-binding experiments to the in vivo situation requires great care. Indeed, iron metabolism in vivo is subject to many factors, some as yet unidentified, that could affect both [Fe.sup.3+] binding and release of free [Fe.sup.2+].
These issues have important implications with respect to both the amount of iron delivered to tissues and the release of redox-active free [Fe.sup.2+], which is an important source of oxidative stress. It is not known whether the increased lipid peroxidation observed in diabetes mellitus is associated with an impairment of [Fe.sup.3+] binding to the two sites on Tf. This hypothesis was suggested by our previous observation that glycation of apoTf led to a decrease in both its TIBC and in its capacity to prevent iron-induced lipid peroxidation (12). In the present study, we aimed to investigate how glycation of Tf affects [Fe.sup.3+] binding on the two sites by analyzing the [Fe.sup.3+]-Tf isoforms of in vitro-glycated Tf. Our results indicate that the effect of glycation on [Fe.sup.3+] binding is dependent on the degree of [Fe.sup.3+] saturation of the Tf molecule. At 32% theoretical [Fe.sup.3+] saturation, the Fe2-Tf isoform was more abundant in the Tf with higher degrees of glycation. Other authors have observed that glycated proteins bind more [Fe.sup.3+] than nonglycated proteins. In the resulting glycochelate complex, however, the metal is more loosely bound and may thus retain redox activity (48). Moreover, glycated holo-Tf (fully [Fe.sup.3+]-saturated Tf) is also known to facilitate the production of free oxygen radicals (49), which can amplify the oxidative effects of iron further.
In contrast, when Tf was almost totally (96%) saturated with [Fe.sup.3+], the proportion of the Fe2-Tf isoform decreased linearly with the degree of glycation. This effect is in accordance with the decrease in TIBC we observed with increasing degrees of glycation (12). The measurement of TIBC is based on the addition of an excess of iron (iron/Tf molar ratio = 2.4). It is known that the antioxidant capacity of Tf (by [Fe.sup.3+] binding) decreases with increasing [Fe.sup.3+] saturation (50). Furthermore, the stability constant for [Fe.sup.3+] is slightly lower for the second binding site (28), and binding of [Fe.sup.3+] leads to rotation of the domains and conformational changes in the Tf molecule (51). This information, together with our observations, implies that the impairment of [Fe.sup.3+] binding by glycation is more apparent at higher [Fe.sup.3+] saturation. The relationship between glycation and [Fe.sup.3+] binding is also seen in the in vivo situation in both rat models and humans. In patients with diabetes, the proportion of glycated Tf is three times higher than in controls (5.2% vs 1.6%) (52) and the TIBC is lower (49,53).
In conclusion, and notwithstanding the analytical shortcomings of the method used to measure [Fe.sup.3+]-Tf isoforms, our results suggest that glycation facilitates the initial binding of [Fe.sup.3+] to Tf but hinders further binding when high amounts of iron are present. In either case, the iron is bound more loosely and is thus more redox-active. This interpretation could explain the decrease in [Fe.sup.3+]-binding antioxidant capacity we observed in glycated apoTf (12). The impairment of Tf function may have important consequences with regard to the appearance of oxidative stress in vivo in diabetes and its involvement in the pathogenesis of diabetic complications. The relationship between the [Fe.sup.3+]-Tf isoforms and glycemia, glycation, oxidative stress, and complication profiles of patients with diabetes requires a more robust method to measure [Fe.sup.3+]-Tf isoforms as well as further investigation in clinical studies. These results could aid in the development of more specific and sensitive techniques capable of identifying patients with a disturbed iron-oxidant-antioxidant balance who are at increased risk of severe diabetic complications.
We thank P. Aerts, M. Vinckx, and J. Vertommen of the Laboratory of Endocrinology for their technical support. This work was supported financially by the Flemish Institute for Scientific-Technological Research.
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ANN VAN CAMPENHOUT,  CHRISTEL VAN CAMPENHOUT,  ALBERT RENE LAGROU,  and BEGONA MANUEL-Y-KEENOY  *
 Laboratory of Endocrinology, Antwerp Metabolic Research Unit, University of Antwerp, Wilrijk, Belgium.
 Laboratory of Immunology and Protein Chemistry, University Hospital of Antwerp, Edegem, Belgium.
 Laboratory of Human Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.
 Nonstandard abbreviations: Tf, transferrin; Fe0-Tf/apoTf, transferrin with no [Fe.sup.3+] ions bound; Fe1N-Tf, monoferric transferrin with [Fe.sup.3+] bound to the N-terminal lobe; Fe1C-Tf, monoferric transferrin with [Fe.sup.3+] bound to the C-terminal lobe; Fe2-Tf, diferric transferrin; IEF, isoelectric focusing; NTA, nitrilotriacetic acid; TIBC, total iron-binding capacity; and NA, neuraminidase.
* Address correspondence to this author at: Laboratory of Endocrinology, Antwerp Metabolic Research Unit, University of Antwerp T 4.37, Universiteitsplein 1, B-2610 Wilrijk, Belgium. Fax 32-3-820-2574; e-mail firstname.lastname@example.org.
Received March 9, 2004; accepted June 3, 2004.
Previously published online at DOI: 10.1373/clinchem.2004.033811
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Van Campenhout, Ann; Van Campenhout, Christel; Lagrou, Albert Rene; Manuel-y-Keenoy, Begona|
|Article Type:||Clinical report|
|Date:||Sep 1, 2004|
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