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 represents a further important parameter for this assessment. The health status of seals is a complex and vague term, 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. Within this context Tf quantities and a varying pattern of its glycoforms are used as indicator for e.g. certain liver diseases, which also 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 regarding the total Tf concentration rather than information of its different glycoforms. Due to a lack of suitable seal Tf antibodies 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 AE 0.03 g L À1(sum of all quantified glycoforms) has been calculated, which is in good agreement with the certified total Tf concentration of 2.35 AE 0.08 g L À1 , confirming the accuracy of the proposed analytical method. Finally, different seal samples were analysed to demonstrate the suitability of the procedure for the quantification of Tf in real samples as well as to observe modified glycoform patterns. Compared to our previous studies for the first time it was possible to quantify the serum Tf baseline reference range for male (1.42-2.35 g L À1) and female German North Sea seals (1.93-2.74 g L À1 ) as well as a CDT level of 0.00-0.10 g L À1 , respectively, which provides valuable further diagnostic information regarding the health status of these specific marine mammals. Compared to assay based quantification approaches the proposed technique indicates great potential to obtain comparable and traceable absolute quantitative results, which are in particular important for long term investigations. This absolute quantification is based on an accurate, traceable element standard, while assay based approaches often show variations depending on the kit quality or changing activities of the used antibodies.

Postmortem Health and Pollution Investigations on Harbor Seals (Phoca vitulina) of the Islands Helgoland and Sylt

“…The Hp concentrations were distinctly elevated for four of five seals (Pv 01, 03, 04, 05) compared to the reference range of harbor seals [19]. The Tf concentrations were elevated for all five seals and the CDT ratio for Pv 01 compared to published reference ranges determined for wildlife harbor seals of the German North Sea [22]. The general higher levels of acute phase proteins measured in this study support the necropsy findings of inflammation, even if individually variations were found (Table S1).…”

“…The measurement of Hp in serum was performed at the HZG using a multispecies Hp assay from Tridelta Development Limited (Maynooth, Kildare, Ireland). The quantification of Tf and CDT was conducted in serum samples as described in former studies at the HZG [20,22].…”

“…The seals used in the present study had to be shot due to severe illness. Using a helicopter transport, the time between death and necropsy was shortened, which helped to maintain the integrity of the samples and to collect sufficient sample amounts for new methodological developments and approaches such as proteomics or in vitro liver cell culture assays [20][21][22][23][24]. The present study provides, for the first time, a basic data set of multiple health parameters and contaminant body burdens of animals from these areas.…”

Kinetics of Iron Import into Developing Mouse Organs Determined by a Pup-swapping Method

“…Indeed, apotransferrin scavenges free iron in the plasma. As long as sufficient apotransferrin is available, virtually all 59 Fe III Cl 3 or 59 Fe III citrate added to plasma will coordinate in minutes (11)(12)(13)(14)(15). These results have been interpreted to mean that virtually all iron in normal plasma is TBI.…”

“…This contradiction stems from uncertainty or disagreement as to the composition, concentration, and stability of NTBI. NTBI might be a population of iron complexes, including heme groups, heme:hemopexin complexes, hemoglobin:haptoglobin complexes, ferritin (9,10,14) and/or iron coordinated to citrate, ascorbate, acetate, or albumin (9, 20 -22).…”