Interaction of the chemotherapeutic agent oxaliplatin and the tyrosine kinase inhibitor dasatinib with the organic cation transporter 2.

Oxaliplatin (OHP) is effective in colorectal cancer treatment but induces peripheral neurotoxicity (OHP-induced peripheral neurotoxicity, OIPN), diminishing survivor quality of life. Organic cation transporter 2 (OCT2) is a key OHP uptake pathway in dorsal root ganglia. Competing for OCT2-mediated OHP uptake, such as with the tyrosine kinase inhibitor dasatinib, may mitigate OHP side effects. We investigated OHP and dasatinib interaction with OCT2 in human embryonic kidney 293 (HEK293) cells expressing OCT2 within a 10-3 to 10-7 M concentration range. Uptake competition experiments using fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+, 1 µM) and mass spectrometry (MS) to determine cellular platinum content indicated that OHP (100 µM) is an OCT2 substrate, mediating OHP cellular toxicity. ASP+ and MS analysis revealed dasatinib as a non-transported inhibitor of hOCT2 (IC50 = 5.9 µM) and as a regulator of OCT2 activity. Dasatinib reduced transporter Vmax, potentially via Y544 phosphorylation suppression. MS analysis showed cellular dasatinib accumulation independent of hOCT2. Although 3 µM dasatinib reduced 100 µM OHP accumulation in hOCT2-HEK293 cells, co-incubation with dasatinib and OHP did not prevent OHP toxicity, possibly due to dasatinib-induced cell viability reduction. In summary, this study demonstrates OHP as an OCT2 substrate and dasatinib as a non-transported inhibitor and regulator of OCT2, offering potential for OIPN mitigation.

Modulating the Activity of the Human Organic Cation Transporter 2 Emerges as a Potential Strategy to Mitigate Unwanted Toxicities Associated with Cisplatin Chemotherapy.

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.

Neutral and Cationic Re(I) Complexes with Pnictogen-Based Coligands and Tunable Functionality: From Phosphorescence to Photoinduced CO Release.

In this work, we have explored Re(I) complexes featuring triphenylpnictogen (PnPh3, Pn = P, As, or Sb)-based coligands and bidentate (neutral or monoanionic) luminophores derived from 1,10-phenantroline (phen), as well as from 2-(3-(tert-butyl)-1H-1,2,4-triazol-5-yl)pyridine (H(N-tBu)). The effect of the increasingly heavy elements on the structural parameters, photoexcited-state properties, and electrochemical behavior as well as the hybridization defects and polarization of the Pn atoms was related to the charges of the main luminophores (i.e., phen vs N-tBu) and explored in terms of photoluminescence spectroscopy, X-ray diffractometry, and quantum-chemical methods. Therefore, an in-depth analysis of the bonding, crystal packing, excited-state energies, and lifetimes was assessed in liquid solutions, frozen glassy matrices, and crystalline phases along with a semiquantitative photoactivation study. Notably, by changing the main ligand from phen to N-tBu, an increase in radiative and radiationless deactivation rates (kr and knr, respectively) at 77 K together with a faster photoinduced CO release and fragmentation at room temperature was detected. In addition, a progressively red-shifted phosphorescence was observed with the growing atomic number of the pnictogen atom, along with a boost in kr and knr at 77 K. Down the Vth main group and upon coordination of the Pn atom to the Re(I) center, an increasingly prominent jump of s-orbital participation on the binding sxp3.00-orbitals of the Pn atoms is evidenced. Based on these findings, the ability of these complexes to act as tunable photoluminescent labels able to perform as light-driven CO-releasing molecules is envisioned.

Quantification of [99Tc]TcO4- in urine by means of anion-exchange chromatography-aerosol desolvation nebulization-inductively coupled plasma-mass spectrometry.

To sensitively determine 99Tc, a new method for internal quantification of its most common and stable species, [99Tc]Tc O 4 - , was developed. Anion-exchange chromatography (IC) was coupled to inductively coupled plasma-mass spectrometry (ICP-MS) and equipped with an aerosol desolvation system to provide enhanced detection power. Due to a lack of commercial Tc standards, an isotope dilution-like approach using a Ru spike and called isobaric dilution analysis (IBDA) was used for internal quantification of 99Tc. This approach required knowledge of the sensitivities of 99Ru and 99Tc in ICP-MS. The latter was determined using an in-house prepared standard manufactured from decayed medical 99mTc-generator eluates. This standard was cleaned and preconcentrated using extraction chromatography with TEVA resin and quantified via total reflection X-ray fluorescence (TXRF) analysis. IC coupled to ICP-MS enabled to separate, detect and quantify [99Tc]Tc O 4 - as most stable Tc species in complex environments, which was demonstrated in a proof of concept. We quantified this species in untreated and undiluted raw urine collected from a patient, who previously underwent scintigraphy with a 99mTc-tracer, and determined a concentration of 19.6 ± 0.5 ng L-1. The developed method has a high utility to characterize a range of Tc-based radiopharmaceuticals, to determine concentrations, purity, and degradation products in complex samples without the need to assess activity parameters of 99(m)Tc.

High-Voltage Instability of Vinylene Carbonate (VC): Impact of Formed Poly-VC on Interphases and Toxicity.

Full exhaustion in specific energy/energy density of state-of-the-art LiNix Coy Mnz O2 (NCM)-based Li-ion batteries (LIB) is currently limited for reasons of NCM stability by upper cut-off voltages (UCV) below 4.3 V. At higher UCV, structural decomposition triggers electrode crosstalk in the course of enhanced transition metal dissolution and leads to severe specific capacity/energy fade; in the worst case to a sudden death phenomenon (roll-over failure). The additive lithium difluorophosphate (LiDFP) is known to suppress this by scavenging dissolved metals, but at the cost of enhanced toxicity due to the formation of organofluorophosphates (OFPs). Addition of film-forming electrolyte additives like vinylene carbonate (VC) can intrinsically decrease OFP formation in thermally aged LiDFP-containing electrolytes, though the benefit of this dual-additive approach can be questioned at higher UCVs. In this work, VC is shown to decrease the formation of potentially toxic OFPs within the electrolyte during cycling at conventional UCVs but triggers OFP formation at higher UCVs. The electrolyte contains soluble VC-polymerization products. These products are formed at the cathode during VC oxidation (and are found within the cathode electrolyte interphase (CEI), suggesting an OFP electrode crosstalk of VC decomposition species, as the OFP-precursor molecules are shown to be formed at the anode.

The role of gadolinium-based contrast agents in magnetic resonance imaging structured reporting and data systems (RADS).

Among the 28 reporting and data systems (RADS) available in the literature, we identified 15 RADS that can be used in Magnetic Resonance Imaging (MRI). Performing examinations without using gadolinium-based contrast agents (GBCA) has benefits, but GBCA administration is often required to achieve an early and accurate diagnosis. The aim of the present review is to summarize the current role of GBCA in MRI RADS. This overview suggests that GBCA are today required in most of the current RADS and are expected to be used in most MRIs performed in patients with cancer. Dynamic contrast enhancement is required for correct scores calculation in PI-RADS and VI-RADS, although scientific evidence may lead in the future to avoid the GBCA administration in these two RADS. In Bone-RADS, contrast enhancement can be required to classify an aggressive lesion. In RADS scoring on whole body-MRI datasets (MET-RADS-P, MY-RADS and ONCO-RADS), in NS-RADS and in Node-RADS, GBCA administration is optional thanks to the intrinsic high contrast resolution of MRI. Future studies are needed to evaluate the impact of the high T1 relaxivity GBCA on the assignment of RADS scores.

Multimodal analytical tools for the molecular and elemental characterisation of lesions in brain tissue of multiple sclerosis patients.

Multiple sclerosis (MS) is a prevalent immune-mediated inflammatory disease of the central nervous system inducing a widespread degradation of myelin and resulting in neurological deficits. Recent advances in molecular and atomic imaging provide the means to probe the microenvironment in affected brain tissues at an unprecedented level of detail and may provide new insights. This study showcases state-of-the-art spectroscopic and mass spectrometric techniques to compare distributions of molecular and atomic entities in MS lesions and surrounding brain tissues. MS brains underwent post-mortem magnetic resonance imaging (MRI) to locate and subsequently dissect MS lesions and surrounding white matter. Digests of lesions and unaffected white matter were analysed via ICP-MS/MS revealing significant differences in concentrations of Li, Mg, P, K, Mn, V, Rb, Ag, Gd and Bi. Micro x-ray fluorescence spectroscopy (µXRF) and laser ablation - inductively coupled plasma - time of flight - mass spectrometry (LA-ICP-ToF-MS) were used as micro-analytical imaging techniques to study distributions of both endogenous and xenobiotic elements. The essential trace elements Fe, Cu and Zn were subsequently calibrated using in-house manufactured gelatine standards. Lipid distributions were studied using IR-micro spectroscopy and matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). MALDI-MSI was complemented with high-resolution tandem mass spectrometry and trapped ion mobility spectroscopy for the annotation of specified phospho- and sphingolipids, revealing specific lipid species decreased in MS lesions compared to surrounding white matter. This explorative study demonstrated that modern molecular and atomic mapping techniques provide high-resolution imaging for relevant bio-indicative entities which may complement our current understanding of the underlying pathophysiological processes.

Contiguous Silver(I)-Mediated Base Pairs of Imidazophenanthroline and Canonical Nucleobases in DNA Duplexes: Formation of Classical Duplexes versus Homodimer Formation.

Metal-mediated base pairs represent a topical alternative to canonical hydrogen-bonded base pairs. In this context, the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was introduced as an artificial nucleobase in a glycol nucleic acid-based nucleoside analogue into a DNA oligonucleotide in a way that the oligonucleotide contains a central block of six contiguous P residues. The ability to engage in Ag+-mediated base pairing was evaluated with respect to the four canonical nucleosides in positions complementary to P. Highly stabilizing Ag+-mediated base pairs were formed with cytosine and guanine (i.e., P-Ag+-C and P-Ag+-G base pairs), whereas the analogous base pairs with thymine and adenine were much less stable and hence formed incompletely. Surprisingly, the intermediate formation of a homodimeric duplex of the P-containing oligonucleotide was observed in all cases, albeit to a different extent. The homodimer is composed of P-Ag+-P base pairs and 18 overhanging mismatched canonical nucleobases. It demonstrates the obstacles present when designing metal-mediated base pairs as metal complexation may take place irrespective of the surrounding natural base pairs. Homodimer formation was found to be particularly prominent when the designated metal-mediated base pairs are of low stability, suggesting that homodimers and regular duplexes are formed in a competing manner.

On-tissue dataset-dependent MALDI-TIMS-MS2 bioimaging.

Trapped ion mobility spectrometry (TIMS) adds an additional separation dimension to mass spectrometry (MS) imaging, however, the lack of fragmentation spectra (MS2) impedes confident compound annotation in spatial metabolomics. Here, we describe spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF), a dataset-dependent acquisition strategy that augments TIMS-MS imaging datasets with MS2 spectra. The fragmentation experiments are systematically distributed across the sample and scheduled for multiple collision energies per precursor ion. Extendable data processing and evaluation workflows are implemented into the open source software MZmine. The workflow and annotation capabilities are demonstrated on rat brain tissue thin sections, measured by matrix-assisted laser desorption/ionisation (MALDI)-TIMS-MS, where SIMSEF enables on-tissue compound annotation through spectral library matching and rule-based lipid annotation within MZmine and maps the (un)known chemical space by molecular networking. The SIMSEF algorithm and data analysis pipelines are open source and modular to provide a community resource.

Combining lipid-mimicking-enabled transition metal and enzyme-mediated catalysis at the cell surface of E. coli.

Being an essential multifunctional platform and interface to the extracellular environment, the cell membrane constitutes a valuable target for the modification and manipulation of cells and cellular behavior, as well as for the implementation of artificial, new-to-nature functionality. While bacterial cell surface functionalization via expression and presentation of recombinant proteins has extensively been applied, the corresponding application of functionalizable lipid mimetics has only rarely been reported. Herein, we describe an approach to equip E. coli cells with a lipid-mimicking, readily membrane-integrating imidazolium salt and a corresponding NHC-palladium complex that allows for flexible bacterial membrane surface functionalization and enables E. coli cells to perform cleavage of propargyl ethers present in the surrounding cell medium. We show that this approach can be combined with already established on-surface functionalization, such as bacterial surface display of enzymes, i.e. laccases, leading to a new type of cascade reaction. Overall, we envision the herein presented proof-of-concept studies to lay the foundation for a multifunctional toolbox that allows flexible and broadly applicable functionalization of bacterial membranes.

Electrochemistry-mass spectrometry bridging the gap between suspect and target screening of valsartan transformation products in wastewater treatment plant effluent.

Degradation of xenobiotics in wastewater treatment plants may lead to the formation of transformation products with higher persistence or increased (eco-)toxic potential compared to the parent compounds. Accordingly, the identification of transformation products from wastewater treatment plant effluents has gained increasing attention. Here, we show the potential of electrochemistry hyphenated to liquid chromatography and mass spectrometry for the prediction of oxidative degradation in wastewater treatment plants using the antihypertensive drug valsartan as a model compound. This approach identifies seven electrochemical transformation products of valsartan, which are used to conduct a suspect screening in effluent of the main wastewater treatment plant in the city of Münster in Germany. Apart from the parent compound valsartan, an electrochemically predicted transformation product, the N-dealkylated ETP336, is detected in wastewater treatment plant effluent. Subsequently, a targeted liquid chromatographytandem mass spectrometry method for the detection of valsartan and its electrochemical transformation products is set up. Here, electrochemical oxidation is used to generate reference materials of the transformation products in situ by hyphenating electrochemistry online to a triple quadrupole mass spectrometer. Using this setup, multiple reaction monitoring transitions are set up without the need for laborious and costly synthesis and isolation of reference materials for the transformation products. The targeted method is then applied to extracts from wastewater treatment plant effluent and the presence of ETP336 and valsartan in the samples is verified. The presented workflow can be used to set up targeted analysis methods for previously unknown transformation products even without the need for expensive high-resolution mass spectrometers.

Species-dependent uptake of gadolinium in Chlamydomonas reinhardtii algae.

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) was used in this study as a valuable tool to assess the species-dependent uptake of metallopharmaceuticals into algal cells. Chlamydomonas reinhardtii algae were incubated for 24 h with four Gadolinium-based contrast agents (GBCAs) and GdCl3. A species dependency towards the uptake of the tested Gd species was observed. Using single cell-ICP-MS, a Gd signal corresponding to single cell events was detected for GdCl3 and the linear GBCAs Omniscan® (Gadodiamide, Gd-DTPA-BMA) and Magnevist® (Gadodiamide, Gd-DTPA). For the macrocyclic complexes Dotarem® (Gadoteric acid, Gd-DOTA) and Gadovist® (Gadobutrol, Gd-BT-DO3A), no such Gd signal was visible. Total Gd analysis via ICP-MS confirmed the presence of Gd in the cells only after incubation with GdCl3 and the linear GBCAs, while only small amounts of Gd were detected for the incubations with macrocyclic GBCAs. Furthermore, the results showed that more Gd is bound to cell structures or macromolecules, while smaller amounts are present in the lysate. Using hydrophilic interaction liquid chromatography (HILIC)-ICP-MS, the soluble Gd species in the lysate were analyzed to determine if the initial Gd complexes were still intact. Surprisingly, no intact GBCAs were detected in the lysates of any incubation solution, possibly due to a change in Gd speciation. Further research is needed to assess which Gd species are present in the lysate, while "free" Gd ions or adducts with cell constituents are the most likely explanation. This study highlights the need for species-dependent investigation of elements in aquatic organisms. Moreover, the uptake of linear GBCAs and their species alteration raises the question of a potential accumulation of Gd in the food chain.

Simultaneous quantification of Gadoxetic acid and Cisplatin in hepatocellular carcinomas using laser ablation-inductively coupled plasma-mass spectrometry.

The gadolinium-based contrast agent Gadoxetic acid and the platinum-based antitumor agent Cisplatin were quantitatively imaged in liver and liver cancer (hepatocellular carcinoma, HCC) tissue of rats by means of laser ablation-inductively coupled plasma-mass spectrometry. HCC bearing rats simultaneously received a tail vein injection of the hepatocyte-specific magnetic resonance imaging contrast agent Gadoxetic acid and a transarterial injection of Cisplatin 15 min before sacrifice and liver removal. Resecting HCC with adjacent liver tissue allows the comparison of Gd, Pt, and endogenous elements like Fe, Cu, and Zn in the various tissue types. Region of interest analysis reveals lower concentrations of Gd in HCC and higher Gd content in the adjacent liver, fitting the selective uptake of Gadoxetic acid into hepatocytes. Furthermore, two malignancy grades and their possible impact on the Gadoxetic acid and Cisplatin uptake are compared. For this, four high grade (G3) and two moderate grade (G2) HCCs were analysed, including a control sample each. Gd concentrations were lower in HCC irrespective of the grade of dedifferentiation (G2, G3) compared to adjacent liver. Despite local arterial Cisplatin injection, concentrations of Pt were similar or also reduced in HCC compared to liver tissue. In addition, endogenous Fe, Cu, and Zn were quantified. While Zn was homogenously distributed, higher Fe concentrations were determined in liver tissue compared to HCC. Hotspots of Cu suggest a deregulated copper homeostasis in certain liver lesions. The Gd and Fe distributions are compared in detail with cellular alterations examined by hematoxylin and eosin staining.

Elemental Bioimaging of Sheep Bone and Articular Cartilage After Single Application of Gadolinium-Based Contrast Agents.

BACKGROUND: Gadolinium-based contrast agents (GBCAs) are applied to enhance magnetic resonance imaging. Gadolinium (Gd), a rare earth metal, is used in a chelated form when administered as GBCA to patients. There is an ongoing scientific debate about the clinical significance of Gd retention in tissues after administration of GBCAs. It is known that bone serves as Gd reservoir, but only sparse information on localization of Gd in bone is available. PURPOSE: The aim of this study was to compare Gd tissue concentration and spatial distribution in femoral epiphysis and diaphysis 10 weeks after single-dose injection of linear and macrocyclic GBCAs in a large animal model. MATERIALS AND METHODS: In this prospective animal study, Swiss-Alpine sheep (n = 36; age range, 4-10 years) received a single injection (0.1 mmol/kg) of macrocyclic (gadobutrol, gadoteridol, and gadoterate meglumine), linear (gadodiamide and gadobenate dimeglumine) GBCAs, or saline. Ten weeks after injection, sheep were killed, and femur heads and shafts were harvested. Gadolinium spatial distribution was determined in 1 sample of each treatment group by laser ablation-inductively coupled plasma-mass spectrometry. All bone specimens were analyzed histopathologically. RESULTS: Injection of GBCAs in female Swiss-Alpine sheep (n = 36) resulted in Gd localization at the endosteal and periosteal surface and in a subset of GBCAs additionally at the cement lines and the bone cartilage junction. No histopathological alterations were observed in the investigated tissue specimens. CONCLUSIONS: Ten weeks after single injection of a clinically relevant dose in adult sheep, both linear species of GBCA resulted in considerably higher accumulation than macrocyclic GBCAs. Gadolinium deposits were restricted to distinct bone and cartilage compartments, such as in bone linings, cement lines, and bone cartilage junctions. Tissue histology remained unaffected.

Laser desorption/ionization-mass spectrometry for the analysis of interphases in lithium ion batteries.

Laser desorption/ionization-mass spectrometry (LDI-MS) is introduced as a complementary technique for the analysis of interphases formed at electrode|electrolyte interfaces in lithium ion batteries (LIBs). An understanding of these interphases is crucial for designing interphase-forming electrolyte formulations and increasing battery lifetime. Especially organic species are analyzed more effectively using LDI-MS than with established methodologies. The combination with trapped ion mobility spectrometry and tandem mass spectrometry yields additional structural information of interphase components. Furthermore, LDI-MS imaging reveals the lateral distribution of compounds on the electrode surface. Using the introduced methods, a deeper understanding of the mechanism of action of the established solid electrolyte interphase-forming electrolyte additive 3,4-dimethyloxazolidine-2,5-dione (Ala-N-CA) for silicon/graphite anodes is obtained, and active electrochemical transformation products are unambiguously identified. In the future, LDI-MS will help to provide a deeper understanding of interfacial processes in LIBs by using it in a multimodal approach with other surface analysis methods to obtain complementary information.

Benfluorex metabolism complemented by electrochemistry-mass spectrometry.

The hypolipidemic and hypoglycemic drug benfluorex was widely applied to treat type 2 diabetes mellitus and metabolic syndrome in overweight patients since 1976. However, benfluorex was connected to multiple cases of valvular heart disease and pulmonary arterial hypertension later on. Similar adverse drug reactions were previously found to be associated to the structurally related drug fenfluramine, which was attributed to the formation of its N-deethylated metabolite norfenfluramine. Even though norfenfluramine was known to be a common metabolite of fenfluramine and benfluorex, only fenfluramine was withdrawn from European and United States markets in 1997 while benfluorex remained available until 2009. In this work, the metabolism of benfluorex is simulated by an online hyphenation of electrochemistry and mass spectrometry and the observed transformation products are further characterized using liquid chromatography and high-resolution tandem mass spectrometry. Using this approach, norfenfluramine is found to be the main electrochemical transformation product of benfluorex. Considering the knowledge about norfenfluramine toxicity, rapid metabolite screening using electrochemistry hyphenated to mass spectrometry could have been used to predict the potential of benfluorex for adverse drug reactions early on, showcasing the value of electrochemical metabolism mimicry for rapid drug safety evaluation.

Analysis of the elemental species-dependent uptake of lanthanide complexes in Arabidopsis thaliana plants by LA-ICP-MS.

Gadolinium-based contrast agents (GBCAs) are found increasingly in different water bodies, making the investigation of their uptake and distribution behavior in plants a matter of high interest to assess their potential effects on the environment. Depending on the used complexing agent, they are classified into linear or macrocyclic GBCAs, with macrocyclic complexes being more stable. In this study, by using TbCl3, Gd-DTPA-BMA, and Eu-DOTA as model compounds for ionic, linear, and macrocyclic lanthanide species, the elemental species-dependent uptake into leaves of Arabidopsis thaliana under identical biological conditions was studied. After growing for 14 days on medium containing the lanthanide species, the uptake of all studied compounds was confirmed by means of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Furthermore, the uptake rate of TbCl3 and the linear Gd-DTPA-BMA was similar, with Tb and Gd hotspots colocated in the areas of hydathodes and the trichomes of the leaves. In contrast, in the case of the macrocyclic Eu-DOTA, Eu was mainly located in the leaf veins. Additionally, Eu was colocated with Tb and Gd in the hydathode at the tip of the leave. Removal of the lanthanide species from the medium led to a decrease in signal intensities, indicating their subsequent release to some extent. However, seven days after the removal, depositions of Eu, Gd, and Tb were still present in the same areas of the leaves as before, showing that complete elimination was not achieved after this period of time. Overall, more Eu was present in the leaves compared to Gd and Tb, which can be explained by the high stability of the Eu-DOTA complex, potentially leading to a higher transport rate into the leaves, whereas TbCl3 and Gd-DTPA-BMA could interact with the roots, reducing their mobility.

Quantitative bioimaging of copper in frozen liver specimens from cats using laser ablation-inductively coupled plasma-mass spectrometry.

OBJECTIVES: The aim of this study was to assess laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) as a tool for measuring concentrations and determining accumulation of copper in frozen liver specimens from cats. METHODS: Six frozen liver specimens were evaluated by qualitative copper staining and quantitative flame atomic absorption spectroscopy. Tissue specimens were cryo-sectioned and quantitative bioimaging of copper was performed using LA-ICP-MS. Results were compared with those obtained using conventional methods. RESULTS: Of the six specimens, only one showed positive staining for copper with rhodanine. Using flame atomic absorption spectroscopy (FAAS), one specimen showed a deficient copper level (<100 µg/g dry weight), two specimens had copper within the reference interval (RI; 150-180 µg/g) and three specimens had copper concentrations above the RI. Bioimaging from LA-ICP-MS showed inhomogeneous distribution of hepatic copper. The areas with dense copper accumulation were represented as hotspots in the liver specimens. Hepatic copper quantification by LA-ICP-MS correlated well with copper quantified by FAAS (r = 0.96, P = 0.002). CONCLUSIONS AND RELEVANCE: Our findings suggest that quantitative bioimaging by LA-ICP-MS could be used to demonstrate the distribution and concentration of copper in frozen liver specimens from cats. The distribution of copper in these specimens was inhomogeneous with dense accumulation represented as hotspots on tissue sections. A positive correlation of hepatic copper concentrations determined by LA-ICP-MS and FAAS was found. Further studies to establish an RI for hepatic copper using this technique and to further determine its clinical utility are warranted.

An automated analysis method enabling the screening of covalent thrombin and factor XIIa inhibitors via liquid chromatography-mass spectrometry.

An automated sample preparation and separation method for the analysis of various enzyme-inhibitor combinations using liquid chromatography (LC) coupled to mass spectrometry (MS) is presented. As conventional anticoagulants have several drawbacks, the most severe being the elevated risk of internal bleedings, it is necessary to develop new-generation anticoagulants with reduced side effects. Therefore, the screening of potential inhibitors against anticoagulation targets like thrombin and FXIIa is important to design a potent and selective inhibitor. To facilitate the analysis of numerous enzyme-inhibitor covalent complexes, automation of the analysis using an LC system with a user-defined injection sequence is helpful. The developed method ensures comparable reaction conditions like reaction time and temperature for all enzyme-inhibitor complexes. Furthermore, it prevents time-consuming manual sample preparation and potential manual errors. To achieve good reproducibility with relative standard deviation of approximately 3% for three-fold determination, multiple cleaning steps were added to the automated sample preparation. Subsequently, this method was applied to screen a variety of 15 aminopyrazole- and aminotriazole-based inhibitors with a covalent mechanism of action against thrombin and to test two covalent inhibitors for FXIIa. Successful complex formation and acylation of the catalytic center of the enzymes was monitored using deconvoluted mass spectra and the matching mass shifts of the acyl moiety of the analyzed inhibitors. The inhibitors' structure directly influenced reaction yields. Sterically demanding aminotriazoles and acyl moieties both affected the product formation negatively. However, the screening yielded several promising candidates for new covalent thrombin inhibitors, which might find their application as prospective anticoagulants.

Electrochemistry/mass spectrometry (EC/MS) for fast generation and identification of novel reactive metabolites of two unsymmetrical bisacridines with anticancer activity.

The development of a new drug requires knowledge about its metabolic fate in a living organism, regarding the comprehensive assessment of both drug therapeutic activity and toxicity profiles. Electrochemistry (EC) coupled with mass spectrometry (MS) is an efficient tool for predicting the phase I metabolism of redox-sensitive drugs. In particular, EC/MS represents a clear advantage for the generation of reactive drug transformation products and their direct identification compared to biological matrices. In this work, we focused on the characterization of novel electrochemical products of two representative unsymmetrical bisacridines (C-2028 and C-2045) with demonstrated high anticancer activity. The electrochemical thin-layer flow-through cell µ-PrepCell 2.0 (Antec Scientific) was used here for the effective metabolite electrosynthesis. The electrochemical simulation of C-2028 reductive and C-2045 oxidative metabolism resulted in the generation of new products that were not observed before. The formation of nitroso [M-O+H]+ and azoxy [2M-3O+H]+ species from C-2028, as well as a series of hydroxylated and/or dehydrogenated products, including possible quinones [M-2H+H]+ and [M+O-2H+H]+ from C-2045, was demonstrated. For the latter, a glutathione S-conjugate (m/z 935.3130) was also obtained in measurements supplemented with the excess of reduced glutathione. For the identification of the products of interest, structural confirmation based on MS/MS fragmentation experiments was performed. Novel products of electrochemical conversions of unsymmetrical bisacridines were discussed in the context of their possible biological effect on the human organism.