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Effects of in Vitro Glycation on Fe³⁺ Binding and Fe³⁺ Isoforms of Transferrin

¹ Laboratory of Endocrinology, Antwerp Metabolic Research Unit, University of Antwerp, Wilrijk, Belgium.
² Laboratory of Immunology and Protein Chemistry, University Hospital of Antwerp, Edegem, Belgium.
³ Laboratory of Human Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.

^aAddress 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 begona.manuelykeenoy{at}ua.ac.be.

Background: In diabetes, protein function is altered by glycation, but the impact on the Fe³⁺ binding and antioxidant functions of transferrin (Tf) is largely unknown. The aim of the present study was to investigate the effects of glycation on the distribution of Fe³⁺ on the two Fe³⁺-binding sites of Tf.

Methods: In vitro glycation of Tf was accomplished by preincubation with glucose for 14 days. Tf was loaded with Fe³⁺ compounds to achieve theoretical Tf Fe³⁺ saturations of 32%, 64%, and 96% (monitored by spectrophotometry). Fe³⁺-Tf isoforms were separated by isoelectric focusing.

Results: Fe³⁺ binding was highest when Tf was incubated with Fe:nitrilotriacetic acid and reached a steady state overnight. Increasing the Fe³⁺ load led to a shift of isoform profile toward the diferric form (Fe2-Tf): in freshly prepared Tf, Fe2-Tf represented 6%, 30%, and 66% of all isoforms at 32%, 64%, and 96% theoretical Fe³⁺ saturation, respectively. Fe³⁺ was equally distributed to the monoferric Tf forms with Fe³⁺ bound to the amino (Fe1N-Tf) and carboxy termini (Fe1C-Tf). Glycation decreased binding of Fe³⁺ to Tf (monitored at 450 nm). At low theoretical Fe³⁺ saturation (32%), glycation increased the mean (SD) proportion of Fe2-Tf: 18 (3)% in the presence of 33.3 mmol/L glucose vs 12 (4)% with 0 mmol/L glucose (P = 0.01). In contrast, at 96% theoretical Fe³⁺ saturation, Fe2-Tf decreased linearly with increasing glycation (r = 0.97; P = 0.008). Preincubation, independent of glycation, favored the Fe1N-Tf isoform at 64% theoretical Fe³⁺ saturation [27 (0.7)% vs 23 (1.1)% of the Fe1C-Tf isoform; P = 0.009].

Conclusions: Glycation impairs Fe³⁺ binding and affects Fe³⁺-Tf isoform distribution depending on concentration. The diagnostic implications of these results need further elucidation in clinical studies.