The Elbe is one of the major rivers releasing pollutants into the coastal areas of the German North Sea. Its estuary represents the habitat of a small population of harbor seals (Phoca vitulina). Only little is known about the health status and contamination levels of these seals. Therefore, a first-ever seal catch was organized next to the islands of Neuwerk and Scharhörn in the region of the Hamburg Wadden Sea National Park. The investigations included a broad set of health parameters and the analysis of metals and organic pollutants in blood samples. Compared to animals of other Wadden Sea areas, the seals showed higher γ-globulin levels, suggesting higher concentrations of pathogens in this near-urban area, elevated concentrations for several metals in particular for V, Sn, Pb, and Sr, and comparable ranges for chlorinated organic contaminants, except for elevated levels of hexachlorobenzene, which indicates characteristic inputs from the Elbe.
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.
The health status of marine mammals such as harbour seals (Phoca vitulina) represents an indirect but powerful way for the assessment of environmental changes. The present work illustrates the first investigation and characterisation of Tf isolated from blood samples of North Sea harbour seals with a view to using changes in Tf isoform patterns as an additional parameter in extended studies of their health status. Therefore, an HPLC-ICP-MS approach has been developed which allows the highly resolved separation and fractionation of up to eight different Tf isoforms, as well as their sensitive and specific detection on the basis of their characteristic iron content. Molecule-specific detection techniques such as nanoLC-ESI-QTRAP-MS or MALDI-TOF-MS were used as complementary techniques to unambiguously identify the isolated proteins as Tf via cross species protein identification and to further characterise the molecular weight as well as the sialic acid content, which is responsible for the elution behaviour of the different isoforms during their ion exchange separation. A molecular mass above 80 kDa has been measured for the different seal Tf isoforms, which is in good agreement with the known molecular mass in other mammalian species, while the estimated pI of the different isoforms indicates some differences in comparison to other species. A number of homologies to known Tf sequences have been observed, which finally allows the cross species protein identification. The combined metallomics orientated analytical approach, which includes the complementary application of element and molecule-specific detection techniques, opens up interesting possibilities for the fast and targeted isolation and identification of a diagnostically relevant metal containing protein from an un-sequenced mammalian species prior to its utilisation in extended studies.
Helgoland and Sylt are important centers of tourism in the North Sea. Harbor and grey seals are one reason for the attraction of these islands. However, little is known about these local seal groups. The present postmortem health and pollution study describes a multiparameter investigation of five ill harbor seals which were shot for animal welfare reasons. Firstly, results of pathology and blood investigations support the bad prognosis of survival made in the field. Signs of inflammation in organs, malnutrition, a high-stress level, and reduced thyroid activity were found. Secondly, metal and organic contaminants were investigated. Metal pollutants in blood, liver, muscle, and kidney tissue were not elevated. Lead and mercury concentrations showed a decreased level compared to former studies. Additionally, interesting insights were found for several organic contaminants in comparison with other studies. The Helgoland seals may be influenced by the contaminants of the Elbe plume.
Marine mammals such as harbour seals (Phoca vitulina) represent a valuable indicator for the state of their habitat or environmental changes in particular due to their key role as top predators within the marine food web. Therefore, the characterization (qualitative and quantitative) of target proteins of such organisms can be of potential use for environmental monitoring processes. In this regard, serum transferrin (Tf) is a key protein related to iron transport and its metabolism. Thus, the present work illustrates the development and comparison of different approaches for the accurate, absolute quantification of Tf isolated from blood samples of North Sea harbour seals with the aim to use possible changes in Tf glycoform patterns as an additional parameter in extended studies, focusing on the assessment of their immune status. For this purpose, different HPLC-ICP-MS approaches have been developed, which allow the highly resolved separation and detection of up to nine different Tf glycoforms in seal blood samples in less than 30 minutes, as well as their sensitive and specific absolute quantification on the basis of their characteristic iron content. One method is based on the application of a reversed gradient sheath flow to compensate gradient related effects during the iron specific detection of Tf. Here a simple flow injection with a certified element standard was used for calibration. The second developed methodology utilizes isotope dilution analysis, which was applied for the quantification of the separated Tf glycoforms. Such accurate protein quantification methods are urgently needed in particular when aiming on comparable long term health related investigations. However, since both methods can be applied independently of the availability of specific protein standards or antibodies they can be seen as universal quantification methods for the targeted biomarker.
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