Bioaccumulation of trace metals in the plastisphere: Awareness of environmental risk from a European perspective.

The term "Plastisphere" refers to the biofilm layer naturally formed by microorganisms attaching to plastic surfaces. This layer possesses the capability to adsorb persistent organic and inorganic pollutants, particularly trace metals, which are the focus of this research study. Immersion experiments were concurrently conducted in five locations spanning four European countries (France, Ireland, Spain, and Italy) utilising eight distinct polymers. These immersions, repeated every three months over a one-year period, aimed to evaluate the baseline bioaccumulation of 12 trace metals. The study underscores the intricate nature of metal bioaccumulation, influenced by both micro-scale factors (such as polymer composition) and macro-scale factors (including geographical site and seasonal variations). Villefranche Bay in France exhibited the lowest metals bioaccumulation, whereas Naples in Italy emerged as the site where bioaccumulation was often the highest for the considered metals. Environmental risk assessment was also conducted in the study. The lightweight nature of certain plastics allows them to be transported across significant distances in the ocean. Consequently, evaluating trace metal concentrations in the plastisphere is imperative for assessing potential environmental repercussions that plastics, along with their associated biota, may exert even in locations distant from their point of emission.

Long-Term Culturing of FreeStyle 293-F Cells Affects Immunoglobulin G Glycome Composition.

Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell "aging". To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG.

Dose imbalance of DYRK1A kinase causes systemic progeroid status in Down syndrome by increasing the un-repaired DNA damage and reducing LaminB1 levels.

BACKGROUND: People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. METHODS: Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. FINDINGS: Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. INTERPRETATION: Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. FUNDING: Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".

A preliminary study of the cultivable microbiota on the plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea, with emphasis on Vibrio isolates and their antibiotic resistance.

Mediterranean Sea is the sixth largest area of marine litter accumulation in the world, and plastic pollution is a growing problem in its Adriatic sub-basin. The aim of the present study was to evaluate the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers in the south-eastern Adriatic Sea in comparison with microbiota in seawater and sediment. Plastic litter in the sea contains an autochthonous microbiota that is different from that of the surrounding seawater and sediment. Vibrio abundance was higher on plastic litter than in surrounding seawater and sediment. All isolated Vibrio showing resistance to ampicillin and vancomycin, while resistance to other antibiotics depended on the isolated species. Overall, this study provides for the first time information on the cultivable microbiota associated with plastic litter collected by commercial fishing trawlers and provides a data base for further studies.

Immunoglobulin G N-Glycosylation Signatures in Incident Type 2 Diabetes and Cardiovascular Disease.

OBJECTIVE: N-glycosylation is a functional posttranslational modification of immunoglobulins (Igs). We hypothesized that specific IgG N-glycans are associated with incident type 2 diabetes and cardiovascular disease (CVD). RESEARCH DESIGN AND METHODS: We performed case-cohort studies within the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort (2,127 in the type 2 diabetes subcohort [741 incident cases]; 2,175 in the CVD subcohort [417 myocardial infarction and stroke cases]). Relative abundances of 24 IgG N-glycan peaks (IgG-GPs) were measured by ultraperformance liquid chromatography, and eight glycosylation traits were derived based on structural similarity. End point-associated IgG-GPs were preselected with fractional polynomials, and prospective associations were estimated in confounder-adjusted Cox models. Diabetes risk associations were validated in three independent studies. RESULTS: After adjustment for confounders and multiple testing correction, IgG-GP7, IgG-GP8, IgG-GP9, IgG-GP11, and IgG-GP19 were associated with type 2 diabetes risk. A score based on these IgG-GPs was associated with a higher diabetes risk in EPIC-Potsdam and independent validation studies (843 total cases, 3,149 total non-cases, pooled estimate per SD increase 1.50 [95% CI 1.37-1.64]). Associations of IgG-GPs with CVD risk differed between men and women. In women, IgG-GP9 was inversely associated with CVD risk (hazard ratio [HR] per SD 0.80 [95% CI 0.65-0.98]). In men, a weighted score based on IgG-GP19 and IgG-GP23 was associated with higher CVD risk (HR per SD 1.47 [95% CI 1.20-1.80]). In addition, several derived traits were associated with cardiometabolic disease incidence. CONCLUSIONS: Selected IgG N-glycans are associated with cardiometabolic risk beyond classic risk factors, including clinical biomarkers.

Immunoglobulin G glycome composition in transition from premenopause to postmenopause.

Gonadal hormones affect immunoglobulin G (IgG) glycosylation, and the more proinflammatory IgG glycome composition might be one of the molecular mechanisms behind the increased proinflammatory phenotype in perimenopause. Using ultra-high-performance liquid chromatography, we analyzed IgG glycome composition in 5,080 samples from 1940 pre-, peri-, and postmenopausal women. Statistically significant decrease in galactosylation and sialylation was observed in postmenopausal women. Furthermore, during the transition from pre- to postmenopausal period, the rate of increase in agalactosylated structures (0.051/yr; 95%CI = 0.043-0.059, p < 0.001) and decrease in digalactosylated (-0.043/yr; 95%CI = -0.050 to -0.037, p < 0.001) and monosialylated glycans (-0.029/yr; 95%CI = -0.034 to -0.024, p < 0.001) were significantly higher than in either pre- or postmenopausal periods. The conversion to the more proinflammatory IgG glycome and the resulting decrease in the ability of IgG to suppress low-grade chronic inflammation may be an important molecular mechanism mediating the increased health risk in perimenopause and postmenopause.

Glycosylation and Aging.

Human lifespan has increased significantly in the last 200 years, emphasizing our need to age healthily. Insights into molecular mechanisms of aging might allow us to slow down its rate or even revert it. Similar to aging, glycosylation is regulated by an intricate interplay of genetic and environmental factors. The dynamics of glycopattern variation during aging has been mostly explored for plasma/serum and immunoglobulin G (IgG) N-glycome, as we describe thoroughly in this chapter. In addition, we discuss the potential functional role of agalactosylated IgG glycans in aging, through modulation of inflammation level, as proposed by the concept of inflammaging. We also comment on the potential to use the plasma/serum and IgG N-glycome as a biomarker of healthy aging and on the interventions that modulate the IgG glycopattern. Finally, we discuss the current knowledge about animal models for human plasma/serum and IgG glycosylation and mention other, less explored, instances of glycopattern changes during organismal aging and cellular senescence.

Rhenium Distribution and Behavior in the Salinity Gradient of a Highly Stratified Estuary and Pristine Riverine Waters (The Krka River, Croatia).

The abundance and distribution of dissolved Re (DRe) were determined in the freshwater part of the Krka River (Croatia), which drains a karst landscape, and in the salinity gradient of its highly stratified estuary. Due to the low DRe concentration, a batch procedure consisting of a pre-concentration step using an anion exchange resin (Dowex) and analysis of DRe in 8 M HNO3 eluate using high-resolution inductively coupled plasma mass spectrometry (HR ICP-MS) was applied. Due to potentially inconsistent recoveries, which ranged from 60 to 87%, quantification was performed using the isotope dilution technique (ID). DRe concentrations in the Krka River increased downstream, from 6.2 pM at the spring site to 11.9 pM upstream of the estuary region. Weathering of the surrounding carbonate lithology is assumed to be the source of the natural Re. Two specific anomalies were registered: a strong increase in DRe concentration due to anthropogenic input near the town of Knin (27.5 pM) and a decrease at a downstream site caused by subsurface input of freshwater from the Zrmanja River, resulting in a relatively low DRe concentration (8.5 pM). In the estuarine region, a near-conservative behavior of DRe was found in the salinity gradient of the upper surface layer, with DRe concentrations ranging from 18 to 38 pM. Anthropogenic input was suspected within the estuarine segment near the urban area, causing a small positive deviation from the conservative line. In the bottom seawater layer, a minor decrease in DRe concentration in the most upstream estuarine regions was apparent, implying weak scavenging of Re.

N-glycosylation of immunoglobulin G predicts incident hypertension.

OBJECTIVES: Glycosylation of immunoglobulin G (IgG) is an important regulator of the immune system and has been implicated in prevalent hypertension. The aim of this study is to investigate whether the IgG glycome begins to change prior to hypertension diagnosis by analysing the IgG glycome composition in a large population-based female cohort with two independent replication samples. METHODS: We included 989 unrelated cases with incident hypertension and 1628 controls from the TwinsUK cohort (mean follow-up time of 6.3 years) with IgG measured at baseline by ultra-performance liquid chromatography and longitudinal BP measurement available. We replicated our findings in 106 individuals from the 10 001 Dalmatians and 729 from KORA S4. Cox regression mixed models were applied to identify changes in glycan traits preincident hypertension, after adjusting for age, mean arterial pressure, BMI, family relatedness and multiple testing (FDR < 0.1). Significant IgG-incident hypertension associations were replicated in the two independent cohorts by leveraging Cox regression mixed models in the 10 001 Dalmatians and logistic regression models in the KORA cohort. RESULTS: We identified and replicated four glycan traits, incidence of bisecting GlcNAc, GP4, GP9 and GP21, that are predictive of incident hypertension after adjusting for confoundes and multiple testing [hazard ratio (95% CI) ranging from 0.45 (0.24-0.84) for GP21 to 2.9 (1.5-5.68) for GP4]. We then linearly combined the four replicated glycans and found that the glycan score correlated with incident hypertension, SBP and DBP. CONCLUSION: Our results suggest that the IgG glycome changes prior to the development of hypertension.

Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study.

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Extensive weight loss reduces glycan age by altering IgG N-glycosylation.

BACKGROUND: Obesity, a major global health problem, is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent posttranslational modification, highly responsive to inflammation and ageing. The prospect of biological age reduction, by changing glycosylation patterns through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort. METHODS: IgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1680 participants from the TwinsUK cohort. RESULTS: Low-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG N-glycome that accompanied progressive weight loss during 1-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG N-glycans (adjusted p value range 7.38 × 10-04-3.94 × 10-02). This IgG N-glycan profile is known to be associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease (adjusted p value 1.79 × 10-02) and an increase in digalactosylation (adjusted p value 5.85 × 10-06). CONCLUSIONS: Altogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age.

Evaluation of diffusive gradients in thin films (DGT) technique for speciation of trace metals in estuarine waters - A multimethodological approach.

Understanding the potential bioavailability of trace metals (TM) in marine systems is of prime importance to implement adapted regulations and efficiently protect our coastal and estuarine waters. In this study Diffusive Gradients in Thin films (DGT) technique with two different pore size was used to evaluate the potentially bioavailable fractions (DGT-labile) of Cd, Co, Cu, Ni, Pb and Zn at various depths of a highly stratified estuary (the Krka River estuary, Croatia) both in winter and summer. DGT-labile concentrations were compared to (1) total dissolved concentrations, (2) concentrations of labile species measured by anodic stripping voltammetry (ASV-labile) for Cu and (3) concentrations derived by chemical speciation modelling. High correlation between dissolved and DGT-labile concentrations was found for all metals, except for Zn where contamination problems prevented reliable conclusions. Percentages of DGT-labile fractions over total dissolved concentrations were (AVG ±â€¯SD): 92 ±â€¯3%, 64 ±â€¯2%, 23 ±â€¯5%, 61 ±â€¯3% and 57 ±â€¯6% for Cd, Pb, Cu, Ni and Co, respectively. No significant difference was found between trace metal concentrations measured with an open pore and restricted pore devices, implying the predominance of kinetically labile metal complexes smaller than 1 nm. For Cu, ASV-labile and DGT labile concentrations were highly correlated (0.97) with ASV-labile concentration being around 35% lower than that of the DGT-labile. Modelling of chemical speciation reliably predicted dynamic (free, inorganic and part of organic complexes) concentration of Cd, whereas dynamic concentrations of Cu and Pb were underestimated by 32% and 65%, respectively. In view of the relative simplicity of DGT devices, they are well suited for the monitoring effort of coastal waters, informing on potentially bioavailable concentrations of TM and thereby, helping to achieve good environmental status of coastal waters, as stipulated within the EU Water Framework Directive.

Organic Copper Speciation by Anodic Stripping Voltammetry in Estuarine Waters With High Dissolved Organic Matter.

The determination of copper (Cu) speciation and its bioavailability in natural waters is an important issue due to its specific role as an essential micronutrient but also a toxic element at elevated concentrations. Here, we report an improved anodic stripping voltammetry (ASV) method for organic Cu speciation, intended to eliminate the important problem of surface-active substances (SAS) interference on the voltammetric signal, hindering measurements in samples with high organic matter concentration. The method relies on the addition of nonionic surfactant Triton-X-100 (T-X-100) at a concentration of 1 mg L-1. T-X-100 competitively inhibits the adsorption of SAS on the Hg electrode, consequently 1) diminishing SAS influence during the deposition step and 2) strongly improving the shape of the stripping Cu peak by eliminating the high background current due to the adsorbed SAS, making the extraction of Cu peak intensities much more convenient. Performed tests revealed that the addition of T-X-100, in the concentration used here, does not have any influence on the determination of Cu complexation parameters and thus is considered "interference-free." The method was tested using fulvic acid as a model of natural organic matter and applied for the determination of Cu speciation in samples collected in the Arno River estuary (Italy) (in spring and summer), characterized by a high dissolved organic carbon (DOC) concentration (up to 5.2 mgC L-1) and anthropogenic Cu input during the tourist season (up to 48 nM of total dissolved Cu). In all the samples, two classes of ligands (denoted as L1 and L2) were determined in concentrations ranging from 3.5 ± 2.9 to 63 ± 4 nM eq Cu for L1 and 17 ± 4 to 104 ± 7 nM eq Cu for L2, with stability constants logK Cu,1 = 9.6 ± 0.2-10.8 ± 0.6 and logK Cu,2 = 8.2 ± 0.3-9.0 ± 0.3. Different linear relationships between DOC and total ligand concentrations between the two seasons suggest a higher abundance of organic ligands in the DOM pool in spring, which is linked to a higher input of terrestrial humic substances into the estuary. This implies that terrestrial humic substances represent a significant pool of Cu-binding ligands in the Arno River estuary.

Improved voltammetric methodology for chromium redox speciation in estuarine waters.

Chromium is a toxic element naturally present in natural waters whose chemical speciation regulates its cycling, mobility and bioavailability. We present here: 1- an improved analytical method for chromium speciation (Cr(VI) vs Cr(III)) in estuarine samples by catalytic adsorptive cathodic stripping voltammetric (cat-AdCSV) and 2- a study highlighting a significant change of redox speciation during summer and winter. Initial measurements first revealed that surface-active substances (SAS) present in estuarine samples strongly influenced the analytical determination of Cr by partially masking the Cr peak through an increase of the background current. We found that the application of a low negative accumulation potential (-1.65 V) resulted in much better voltammograms compared to those obtained using the usual accumulation potential of -1.0 V. Using humic acid (HA) as a model SAS of natural origin, we show that this negative potential clearly prevents adsorption of SAS on the Hg-electrode surface, which in turns benefits the adsorption of the in-situ formed Cr(III)-DTPA complex and the resulting signal. The optimised method was applied to determine chromium redox speciation and distribution along the 23 km long salinity gradient, well oxygenated, Krka River estuary (Croatia). Cr(VI) was found to be the dominant redox species in both summer and winter, with Cr(III) contribution being lower in summer (up to ∼30%, average of ∼5%) than in winter (up to ∼50%, average of ∼30%). In summer, lower concentrations of Cr(VI) were found in the freshwater end-member (2.5 nM) than in the seawater end-member (4-5 nM), while the opposite trend was found in winter. Hexavalent chromium exhibited a non-conservative behaviour along the salinity gradient for both seasons. Chromium predominantly exists in dissolved phase, and contribution of particles reactive Cr(III) was minor.

Trace Metals in Noah's Ark Shells (Arca noae Linnaeus, 1758): Impact of Tourist Season and Human Health Risk.

Commercially important bivalve Noah's Ark shell (Arca noae Linnaeus, 1758) represents a high-quality seafood product, but the data on levels of metal contaminants that could pose a human health risk and also on some essential elements that are important for health protection are lacking. This study examined the concentrations of Cd, Pb, Cr, Ni, Cu, Co, and Zn in the soft tissue of A. noae from harvesting area in the central Adriatic Sea, to survey whether heavy metals are within the acceptable limits for public health and whether tourism could have an impact on them. The concentrations of analysed metals varied for Cd: 0.15-0.74, Pb: 0.06-0.26, Cr: 0.11-0.34, Ni: 0.09-0.22, Cu: 0.65-1.95, Co: 0.04-0.09, and Zn: 18.3-74.7 mg/kg wet weight. These levels were lower than the permissible limits for safe consummation of seafood, and only for Cd, some precautions should be taken into account if older shellfish were consumed. Increase of Cd, Cr, and Cu in shell tissue was observed during the tourist season at the site closest to the marine traffic routes, indicating that metal levels in shellfish tissue should be monitored especially carefully during the peak tourist season to prevent eventual toxic effects due to increased intake of metals, specifically of Cd.

Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary: The Krka River estuary (Adriatic, Croatia).

Distributions of trace metals (TM), organic carbon, SPM and physico-chemical parameters were studied in the highly stratified Krka River estuary in winter/summer periods. The non-conservative behaviour of Zn, Cd, Pb and Cu in the brackish layer (plume), easily spotted due to very low inputs by the river, was mainly caused by their inputs from the pleasure boats, nautical marinas and harbour (e.g. release from antifouling paints). Contrarily, Ni and Co followed near-conservative behaviour. The extremely low SPM discharged by the river, resulted in a predominant dissolved fraction (>80%) of all TM, except Pb. Vertical scavenging, coupled with the long residence time, caused accumulation and progressive upstream increase of TM and SPM in the bottom seawater. Decrease of distribution coefficient (KD) in the brackish layer for winter period was ascribed to the change of SPM nature (terrestrial vs. biogenic), whereas a variable and increased biogenic component of SPM caused scattered KDs in summer.

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules.