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Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status.
Manganese (Mn) and zinc (Zn) are not only essential trace elements, but also potential exogenous risk factors for various diseases. Since the disturbed homeostasis of single metals can result in detrimental health effects, concerns have emerged regarding the consequences of excessive exposures to multiple metals, either via nutritional supplementation or parenteral nutrition. This study focuses on Mn-Zn-interactions in the nematode Caenorhabditis elegans (C. elegans) model, taking into account aspects related to aging and age-dependent neurodegeneration.
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C. elegans
(2015)
Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology.
The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid+ chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation
Manganese (Mn) is an essential micronutrient for development and function of the nervous system. Deficiencies in Mn transport have been implicated in the pathogenesis of Huntington's disease (HD), an autosomal dominant neurodegenerative disorder characterized by loss of medium spiny neurons of the striatum. Brain Mn levels are highest in striatum and other basal ganglia structures, the most sensitive brain regions to Mn neurotoxicity. Mouse models of HD exhibit decreased striatal Mn accumulation and HD striatal neuron models are resistant to Mn cytotoxicity. We hypothesized that the observed modulation of Mn cellular transport is associated with compensatory metabolic responses to HD pathology. Here we use an untargeted metabolomics approach by performing ultraperformance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS) on control and HD immortalized mouse striatal neurons to identify metabolic disruptions under three Mn exposure conditions, low (vehicle), moderate (non-cytotoxic) and high (cytotoxic). Our analysis revealed lower metabolite levels of pantothenic acid, and glutathione (GSH) in HD striatal cells relative to control cells. HD striatal cells also exhibited lower abundance and impaired induction of isobutyryl carnitine in response to increasing Mn exposure. In addition, we observed induction of metabolites in the pentose shunt pathway in HD striatal cells after high Mn exposure. These findings provide metabolic evidence of an interaction between the HD genotype and biologically relevant levels of Mn in a striatal cell model with known HD by Mn exposure interactions. The metabolic phenotypes detected support existing hypotheses that changes in energetic processes underlie the pathobiology of both HD and Mn neurotoxicity.
Background: Transport of methylmercury (MeHg) across the blood-brain barrier towards the brain side is well discussed in literature, while ethylmercury (EtHg) and inorganic mercury are not adequately characterized regarding their entry into the brain. Studies investigating a possible efflux out of the brain are not described to our knowledge.
Methods: This study compares, for the first time, effects of organic methylmercury chloride (MeHgCl), EtHg-containing thiomersal and inorganic Hg chloride (HgCl2) on as well as their transfer across a primary porcine in vitro model of the blood-brain barrier.
Results: With respect to the barrier integrity, the barrier model exhibited a much higher sensitivity towards HgCl2 following basolateral incubation (brain-facing side) as compared to apical application (blood-facing side). These HgCl2 induced effects on the barrier integrity after brain side incubation are comparable to that of the organic species, although MeHgCl and thiomersal exerted much higher cytotoxic effects in the barrier building cells. Hg transfer rates following exposure to organic species in both directions argue for diffusion as transfer mechanism. Inorganic Hg application surprisingly resulted in a Hg transfer out of the brain-facing compartment.
Conclusions: In case of MeHgCl and thiomersal incubation, mercury crossed the barrier in both directions, with a slight accumulation in the basolateral, brain-facing compartment, after simultaneous incubation in both compartments. For HgCl2, our data provide first evidence that the blood-brain barrier transfers mercury out of the brain.
The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.
Zinc deficiency has a fundamental influence on the immune defense, with multiple effects on different immune cells, resulting in a major impairment of human health. Monocytes and macrophages are among the immune cells that are most fundamentally affected by zinc, but the impact of zinc on these cells is still far from being completely understood. Therefore, this study investigates the influence of zinc deficiency on monocytes of healthy human donors. Peripheral blood mononuclear cells, which include monocytes, were cultured under zinc deficient conditions for 3 days. This was achieved by two different methods: by application of the membrane permeable chelator N,N,N0´,N0´-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) or by removal of zinc from the culture medium using a CHELEX 100 resin. Subsequently, monocyte functions were analyzed in response to Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Zinc depletion had differential effects. On the one hand, elimination of bacterial pathogens by phagocytosis and oxidative burst was elevated. On the other hand, the production of the inflammatory cytokines tumor necrosis factor (TNF)-a and interleukin (IL)-6 was reduced. This suggests that monocytes shift from intercellular communication to basic innate defensive functions in response to zinc deficiency. These results were obtained regardless of the method by which zinc deficiency was achieved. However, CHELEX-treated medium strongly augmented cytokine production, independently from its capability for zinc removal. This side-effect severely limits the use of CHELEX for investigating the effects of zinc deficiency on innate immunity.
Soils in Germany are commonly low in selenium; consequently, a sufficient dietary supply is not always ensured. The extent of such provision adequacy is estimated by the optimal effect range of biomarkers, which often reflects the physiological requirement. Preceding epidemiological studies indicate that low selenium serum concentrations could be related to cardiovascular diseases. Inter alia, risk factors for cardiovascular diseases are physical inactivity, overweight, as well as disadvantageous eating habits. In order to assess whether these risk factors can be modulated, a cardio-protective diet comprising fixed menu plans combined with physical exercise was applied in the German MoKaRi (modulation of cardiovascular risk factors) intervention study. We analyzed serum samples of the MoKaRi cohort (51 participants) for total selenium, GPx activity, and selenoprotein P at different timepoints of the study (0, 10, 20, 40 weeks) to explore the suitability of these selenium-associated markers as indicators of selenium status. Overall, the time-dependent fluctuations in serum selenium concentration suggest a successful change in nutritional and lifestyle behavior. Compared to baseline, a pronounced increase in GPx activity and selenoprotein P was observed, while serum selenium decreased in participants with initially adequate serum selenium content. SELENOP concentration showed a moderate positive monotonic correlation (r = 0.467, p < 0.0001) to total Se concentration, while only a weak linear relationship was observed for GPx activity versus total Se concentration (r = 0.186, p = 0.021). Evidently, other factors apart from the available Se pool must have an impact on the GPx activity, leading to the conclusion that, without having identified these factors, GPx activity should not be used as a status marker for Se