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

Abstract

Harbour seals (Phoca vitulina) are bio indicators for the assessment of their habitat and environmental changes. Besides population parameters and trends (survival, age structure, sex ratio), the individual health status is an important parameter for this assessment. The health status of seals is a complex and vague item, determined by a wide range of diagnostic parameters. Quantities of important blood proteins such as transferrin (Tf), as well as altered distribution patterns of its glycoforms, are frequently used as biomarkers in clinical diagnosis. Here Tf quantities and varying pattern of its glycoforms are used as indicator for e.g. a general impairment of the liver as well as certain liver diseases, which represents one of the most frequently observed pathological indication in harbour seals of the North Sea. Currently, most assay based quantification methods for Tf are limited since they often provide only information of the total Tf concentration rather than its different isoforms or deliver only relative concentrations of the Tf isoforms. Due to the lack of suitable antibodies for seal Tf also the application of more specific antibody based approaches is not possible. Within this background a new approach for the absolute quantification of the iron-transport protein Tf in the blood of harbour seals using its characteristic iron content and HPLC-ICP-MS detection is described. Method validation was performed using a certified human serum reference material (ERM-DA470K/IFCC). A Tf concentration of 2.33 ± 0.03 g L-1 (sum of all quantified isoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 ± 0.08 g L-1, confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the method for the quantification of Tf in real samples as well as to observe modified glycoform patterns. A baseline reference range of 1.50 – 2.44 g L-1 Tf in serum and 0.00 – 0.18 g L-1 CDT in serum of harbour seals of the German North Sea was estimated.