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Multi-element analysis in cytosolic fractions in selected biological samples using HR ICP-MS

The common approach of measuring only the total concentrations of macro- and trace elements in biological materials does not provide complete information about their bioavailability and toxicity, which is mostly associated with their binding to essential molecules in the cytosol [1]. The aim of this study was to apply and validate multielement analysis in the cytosolic fractions of northern pike (Esox lucius) liver and Unio crassus digestive gland, based on the procedure previously described for the analysis of several metals in the hepatic cytosol of European chub (Squalius cephalus) [2]. After tissue homogenization, taking in consideration the specificities of each tissue, the homogenates were centrifuged at 50.000×g for 2 hours at 4 °C and the supernatants (cytosolic fractions containing metabolically active proteins and microsomes [3]) were obtained. An oxidation mixture containing concentrated HNO3 and 30% H2O2 (v/v 3:1) was added to the cytosolic fractions (v/v 1:1). Digestion was performed in a laboratory drying oven at 85 °C for 3.5 h. Thereafter, the samples were diluted with Mili-Q water, five times for Ca/trace element analysis and 20 times for Na/K/Mg analysis on HR ICP-MS. Indium was added to all samples as an internal standard. Appropriate dilutions of the multi-element stock standard solution in 2% HNO3 were used for external calibration. The method was validated by determining the limits of detection (LOD) and quantification (LOQ) for 30 elements (26 trace and four macro), the repeatability of measurement in duplicates, and measurement accuracy based on analysis of quality control samples. Out of 30 analyzed elements, the method enabled the reliable measurement of 19 elements in the E. lucius liver and of 27 elements in the U. crassus digestive gland. The study demonstrated that a high number of elements, both essential and highly toxic nonessential elements, can be accurately measured in the digested cytosols of fish liver and mussels digestive glands using ICP- MS. Metal quantification in the cytosolic fraction containing metalloproteins could provide more information about the biological effects of metals than a study limited to metal concentrations in whole tissues.

multi-element ; cytosol ; fish ; mussel ; HR ICP-MS

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