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

Abstract

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.

Increased serotransferrin and ceruloplasmin turnover in diet-controlled patients with type 2 diabetes

“…Since Trf glycation and Cp deamidation may affect their biological functions [4,5], we measured serum iron and copper levels, Cp enzymatic activity, Trf iron saturation, TIBC, UIBC, and the extent of systemic oxidative stress as surrogates to Trf and Cp function. Our results did not show any loss in Trf iron-binding capacity and iron saturation in T2DM patients.…”

“…The Fe 2+ released from enterocytes, macrophages and hepatocytes is oxidized to Fe 3+ by plasma ferroxidases, such as hephaestin and ceruloplasmin (Cp), for incorporation into serotransferrin (Trf), which delivers iron to all cells [3]. The specific interaction between Cp and Trf with iron depends on the structural integrity of these proteins that is critical to iron mobilization and transport [4,5]. …”

Surgical Resection and Liposomal Amphotericin B to Treat Cavitary Pulmonary Zygomycosis in a Patient With Diabetes

“…Also the iron-binding capacity of normal serum decreases in acidotic conditions and with glycosylation of transferrin, as happens in diabetic patients. 6 Patients on deferoxamine are particularly vulnerable to zygomycetes. The symptoms of zygomycosis are highly variable and depend on the affected sites and the extent of the disease.…”

“…The pulmonary form of zygomycosis is difficult to diagnose because of the nonspecific radiological findings and the usual necessity of an invasive approach and biopsy. 6 An early multidisciplinary approach is critical for successful treatment of zygomycosis. It is necessary to reverse the predisposing condition whenever possible, to surgically debride devitalized tissue, and to administer appropriate antifungal therapy.…”

Absolute quantification of transferrin in blood samples of harbour seals using HPLC-ICP-MS

“…For human serotransferrin of healthy individuals the saturation is about 30%. 25,26 The known iron stoichiometry as well as the high stability constant of the Tf-Fe complex (ranging from 10 À19 -10 À22 M À1 for human Tf as specified in the literature [27][28][29][30] ) facilitates the application of sensitive element specific detection methods, such as inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to HPLC for the separation and detection of this protein. 23,31 Del Castillo Busto demonstrates the application of iron isotope dilution analysis (IDA) for the quantification of human carbohydrate deficient transferrin (CDT), which is considered as a reliable marker for chronic alcohol abuse.…”