Early Stage

8. Development of an HPLC-based Method for the Separation of Transferrin Forms in Murine Serum

Introduction: Transferrin (Tf) is the primary iron binding protein in serum. It has two homologous lobes, N and C, that can each bind one iron molecule. Thus, Tf exists in four forms, apo-, monoferric N, monoferric C and holo-Tf. At physiologic Tf saturations, the monoferric Tfs are the predominant forms. Using mice genetically engineered to block iron binding in either the N or C lobe of Tf, we have shown that the monoferric Tfs differentially influence erythropoietin (Epo) responsiveness depending on which lobe contains iron.  Anemia is a common comorbidity in chronic kidney disease (CKD). It is characterized by Epo hypo-responsiveness, which may be influenced by the specific lobe distribution of iron in Tf. Clinical analyses to investigate this possibility are pending. However, large-scale investigation has been difficult because only suboptimal techniques are available to separate the Tf isoforms from serum. We developed and are in the process of validating an HPLC-based assay for the separation of the four Tf forms from human serum. Parallel investigations using mouse models would be ideal. However, the methodology utilized to separate human Tfs does not provide adequate resolution for mouse Tfs. We hypothesize that murine Tf forms can be adequately resolved with modification of the HPLC assay developed for resolution of human isoforms.

Methods: To test this hypothesis, we investigated the effects of altering the pH of Buffer A in a strong anion exchange HPLC protocol on the resolution of the murine Tf forms.

Results: Results indicate progressively longer retention times with increasing pH, with improved resolution at pH 7.5 producing four discernable peaks corresponding to the four forms of Tf in mouse serum. Tentative peak identification has been assigned by comparing the retention times of the Tfs with serum from wild-type and each of the monoferric mutant mice. Although increased pH improves resolution, additional optimization is required for full resolution of peaks.

Impact: Once achieved, this assay will provide a technique to investigate the influence of the monoferric Tfs on pathology in murine models of CKD and other conditions characterized by alterations in Epo responsiveness.

Organization – Saint Louis University

Parrow NL, Violet PC, George NA, Levine M, Fleming RE