Size-exclusion HPLC analysis of trace element distributions in hepatic and gill cytosol of Vardar chub (Squalius vardarensis Karaman) from mining impacted rivers in north-eastern Macedonia

Cytosolic Distribution of Metals (Cd, Cu) and Metalloids (As, Se) in Livers and Gonads of Field-Collected Fish Exposed to an Environmental Contamination Gradient: An SEC-ICP-MS Analysis

“…In 1991, Klaverkamp et al [12] reported that in white suckers collected from a metal-contaminated environment, half of the hepatic Cu burden was bound to MT. Our results showed that Cu distribution in white suckers agreed with previous studies investigating Cu-binding biomolecules in the liver of freshwater fish collected in metal-impacted areas, where most of Cu was present at the same elution time as the elution time for MT (European eels (Anguilla anguilla) [13,14]; European chub and Vardar chub (Squalius cephalus and S. vardarensis) [15,16]; gibel carp (Carassius auratus gibelio) [17]). Comparable interpretations were made in field-collected juvenile yellow perch (Perca flavescens), although the major Cu peak did not elute at exactly the same elution time as was observed for the MT standard [18]; many isoforms of MTs co-exist and may explain this difference [17].…”

“…Co-elution with Cu was also reported, and the authors speculated that the metal-binding ligand was probably MT. Cadmium presence in the cytosol and its probable binding to MT were previously reported for the liver and gills of Vardar and European chubs collected in metal mining-impacted rivers [15,16,19]. Increased Cd associated with hepatic MT was also reported in field-collected European eels (Anguilla anguilla) and gibel carp (Carassius auratus gibelio) [14,17].…”

Characterization and identification of selected metal-binding biomolecules from hepatic and gill cytosols of Vardar chub (Squalius vardarensis Karaman, 1928) using various techniques of liquid chromatography and mass spectrometry

“…In our previous work we have implemented offline coupling of SEC-HPLC and high resolution (HR) ICP-MS to determine distributions of several elements (Cd, Co, Cu, Fe, Mn, Mo, Pb, Se, Tl and Zn) among cytosolic biomolecules of the gills and/or liver of three fish species, 4 European chub (Squalius cephalus) [27][28] , Vardar chub (Squalius vardarensis) 29 and brown trout (Salmo trutta) 30 . In our studies, the liver were chosen as a key metabolic and detoxification organ of fish, containing a high amount of various metalloproteins which are characterized by a wide range of molecular masses 7,31 .…”

“…The resolution and sensitivity of SEC-HPLC method is such that it offers a rough separation of studied metal-binding biomolecules, and obtained elution peaks of each studied element generally cover rather wide ranges of molecular masses, which can include several metalloproteins. Furthermore, elution peaks of different metals can partially or completely overlap, thus needing a further separation before any kind of additional analyses [27][28][29][30] .…”

“…Hence, in this study, which presents the continuation of our previous study on the liver and the gills of Vardar chub 29 , our first goal was to implement anion-exchange chromatography as an additional separation technique following the size-exclusion chromatography, to isolate purified fractions that contain, whenever possible, individual metal-binding biomolecules. Our second goal was to implement two mass spectrometry techniques into our research (MALDI-TOF-MS and LC-MS/MS), to more precisely determine the molecular masses of several metal-binding biomolecules from hepatic and gill cytosols of Vardar chub, and, when possible, to identify them.…”