Christoph A. Wehe

Diagnosis of Nephrogenic Systemic Fibrosis by means of Elemental Bioimaging and Speciation Analysis

The combined use of elemental bioimaging and speciation analysis is presented as a novel means for the diagnosis of nephrogenic systemic fibrosis (NSF), a rare disease occurring after administration of gadolinium-based contrast agents (GBCA) for magnetic resonance imaging (MRI), in skin samples of patients suffering from renal insufficiency. As the pathogenesis of NSF is still largely unknown particularly with regard to the distribution and potential retention of gadolinium in the human organism, a skin biopsy sample from a suspected NSF patient was investigated. The combination of inductively coupled plasma mass spectrometry (ICP-MS), laser ablation (LA) ICP-MS for quantitative elemental bioimaging, and hydrophilic interaction liquid chromatography (HILIC) ICP-MS for speciation analysis allowed one to unambiguously diagnose the patient as a case of NSF. By means of ICP-MS, a total gadolinium concentration from 3.02 to 4.58 mg/kg was determined in the biopsy sample, indicating a considerable deposition of gadolinium in the patients skin. LA-ICP-MS revealed a distinctly inhomogeneous distribution of gadolinium as well as concentrations of up to 400 mg/kg in individual sections of the skin biopsy. Furthermore, the correlation between the distributions of phosphorus and gadolinium suggests the presence of GdPO4 deposits in the tissue section. Speciation analysis by means of HILIC-ICP-MS showed the presence of the intact GBCA Gd-HP-DO3A eight years after the administration to the patient. The concentration of the contrast agent in the aqueous extract of the skin biopsy was found to be 1.76 nmol/L. Moreover, evidence for the presence of further highly polar gadolinium species in low concentrations was found.

Quantitative bioimaging of platinum in polymer embedded mouse organs using laser ablation ICP-MS

A novel quantification approach for tissue imaging using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) based on tissue embedding in cold-curing resins (Technovit 7100) is presented. With respect to massive side effects on cisplatin, the platinum distribution at different time intervals after cisplatin treatment of mice was determined quantitatively in different tissues including cochlea, testis and kidney. For this purpose, cold-curing resin blocks spiked with different amounts of platinum acetyl acetonate prior to curing were ablated after sectioning at 5 μm thickness and were analysed using ICP-MS after microwave digestion. High spatial resolution and limits of detection in the low ppb range (8 μg kg(-1)) were achieved using a simple and efficient sample preparation. External calibration using the Technovit 7100 standards proved to yield precise and reproducible quantification results. The distribution and retention behaviour of cisplatin in the organs was investigated using the new calibration method.

Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro

Background: Modes of neurotoxic action after Mn overexposure are not fully understood. Results: The blood-CSF barrier showed higher Mn sensitivity and active Mn transport properties. Conclusion: The blood-CSF barrier might be the major route for Mn into the brain. Significance: Deeper insight in the impact of Mn on and its transfer across brain barrier systems might help to prevent irreversible neurological damage. Manganese occupational and dietary overexposure has been shown to result in specific clinical central nervous system syndromes, which are similar to those observed in Parkinson disease. To date, modes of neurotoxic action of Mn are still to be elucidated but are thought to be strongly related to Mn accumulation in brain and oxidative stress. However, the pathway and the exact process of Mn uptake in the brain are yet not fully understood. Here, two well characterized primary porcine in vitro models of the blood-brain and the blood-cerebrospinal fluid (CSF) barrier were applied to assess the transfer of Mn in the brain while monitoring its effect on the barrier properties. Thus, for the first time effects of MnCl2 on the integrity of these two barriers as well as Mn transfer across the respective barriers are compared in one study. The data reveal a stronger Mn sensitivity of the in vitro blood-CSF barrier compared with the blood-brain barrier. Very interestingly, the negative effects of Mn on the structural and functional properties of the highly Mn-sensitive blood-CSF barrier were partly reversible after incubation with calcium. In summary, both the observed stronger Mn sensitivity of the in vitro blood-CSF barrier and the observed site-directed, most probably active, Mn transport toward the brain facing compartment, reveal that, in contrast to the general assumption in literature, after oral Mn intake the blood-CSF barrier might be the major route for Mn into the brain.

Sensitive quantification of gadolinium-based magnetic resonance imaging contrast agents in surface waters using hydrophilic interaction liquid chromatography and inductively coupled plasma sector field mass spectrometry.

The application of gadolinium(Gd)-based contrast agents to support medical examinations by magnetic resonance imaging (MRI) results in a large input of Gd into the environment. The long-term effects of the anthropogenic Gd anomaly, especially on aqueous ecosystems, are mostly unknown. The identification and quantification of Gd-based contrast agents in the aquatic environment requires the use of powerful methods of speciation analysis. Therefore, a method employing the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma sector field mass spectrometry (ICP-SFMS) with sample introduction as dry aerosol generated by desolvation was developed. The desolvation resulted in improved limits of detection for the predominantly used contrast agents well below 0.10 nmol/L. This method was subsequently used for the analysis of Gd species in surface waters. Samples from a nature reserve in the city of Münster (Germany), into which the effluent from the citys main wastewater treatment plant enters the environment, were examined. The contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were identified and quantified in constant ratios in those samples. The concentrations were found in a range from 0.59 nmol/L for Gd-DOTA up to 3.55 nmol/L for Gd-BT-DO3A. As a result of mass balancing, the contrast agent concentration was found to account for 74-89% of total Gd concentrations, possibly indicating the presence of further Gd species. Nevertheless, there was no direct indication of species transformation by transmetallation reactions resulting in such Gd species. The determination of REE patterns by means of ICP-MS confirmed the results of speciation analysis showing significant Gd anomalies.

Speciation and isotope dilution analysis of gadolinium-based contrast agents in wastewater.

The fate of Gadolinium (Gd)-based contrast agents for magnetic resonance imaging (MRI) during sewage treatment was investigated. The total concentration of Gd in influent and effluent 2 and 24 h composite samples was determined by means of isotope dilution analysis. The balancing of Gd input and output of a sewage plant over seven days indicated that approximately 10% of the Gd is removed during treatment. Batch experiments simulating the aeration tank of a sewage treatment plant confirmed the Gd complex removal during activated sludge treatment. For speciation analysis of the Gd complexes in wastewater samples, high performance liquid chromatography (HPLC) was hyphenated to inductively coupled plasma sector field mass spectrometry (ICP-SFMS). Separation of the five predominantly used contrast agents was carried out on a new hydrophilic interaction liquid chromatography stationary phase in less than 15 min. A limit of detection (LOD) of 0.13 μg/L and a limit of quantification of 0.43 μg/L could be achieved for the Gd chelates without having to apply enrichment techniques. Speciation analysis of the 24 h composite samples revealed that 80% of the Gd complexes are present as Gd-BT-DO3A in the sampled treatment plant. The day-of-week dependent variation of the complex load followed the variation of the total Gd load, indicating a similar behavior. The analysis of sewage sludge did not prove the presence of anthropogenic Gd. However, in the effluent of the chamber filter press, which was used for sludge dewatering, two of the contrast agents and three other unknown Gd species were observed. This indicates that species transformation took place during anaerobic sludge treatment.

Elemental bioimaging of haematoxylin and eosin-stained tissues by laser ablation ICP-MS

Haematoxylin and eosin are frequently used histological stains (“H & E-stains”) for the visualization of tissue structures, which is particularly important for the diagnosis of various diseases including cancer. These stains contain aluminum and bromine in their chemical structures, thus providing access for their visualization by means of ICP-MS imaging. Different tissues including appendix, lymph nodes, Fallopian tube and esophageal tumor were investigated. By means of this novel approach, orthogonal data in comparison to optical micrographs, commonly used by the pathologist, were obtained. Hence, different entities of the parallel or serial tissue sections can now be allocated to the manifold set of elemental information acquired by LA-ICP-MS. In surgically removed and H & E-stained human tissues, the images of aluminum and bromine with resolution down to 10 μm were generated. In addition, elemental distribution maps of carbon, aluminum, bromine and platinum in unstained and stained human esophageal tumor sections after Cisplatin therapy were generated, showing a further expanding the capabilities of this technique. As both stained and unstained samples showed a similar platinum distribution, integrity of the platinum distribution after the staining process could be verified. In addition, no significant background of aluminum or bromine in unstained samples was observed, showing the successful reduction of polyatomic, isobaric interferences by utilization of a collision/reaction cell.

Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis

RATIONALE Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research.

Elemental bioimaging of nanosilver-coated prostheses using X-ray fluorescence spectroscopy and laser ablation-inductively coupled plasma-mass spectrometry.

The distribution of different chemical elements from a nanosilver-coated bone implant was visualized, combining the benefits of two complementary methods for elemental bioimaging, the nondestructive micro X-ray fluorescence (μ-XRF), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Challenges caused by the physically inhomogeneous materials including bone and soft tissues were addressed by polymer embedding. With the use of μ-XRF, fast sample mapping was achieved obtaining titanium and vanadium signals from the metal implant as well as phosphorus and calcium signals representing hard bone tissue and sulfur distribution representing soft tissues. Only by the use of LA-ICP-MS, the required high sensitivity and low detection limits for the determination of silver were obtained. Metal distribution within the part of cancellous bone was revealed for silver as well as for the implant constituents titanium, vanadium, and aluminum. Furthermore, the detection of coinciding high local zirconium and aluminum signals at the implant surface indicates remaining blasting abrasive from preoperative surface treatment of the nanosilver-coated device.

In vitro intestinal bioavailability of arsenosugar metabolites and presystemic metabolism of thio-dimethylarsinic acid in Caco-2 cells

Two arsenosugar metabolites show in vitro intestinal bioavailability similar to that of arsenite, and much higher than that of arsenosugars.

Tracing gadolinium-based contrast agents from surface water to drinking water by means of speciation analysis.

In recent decades, a significant amount of anthropogenic gadolinium has been released into the environment as a result of the broad application of contrast agents for magnetic resonance imaging (MRI). Since this anthropogenic gadolinium anomaly has also been detected in drinking water, it has become necessary to investigate the possible effect of drinking water purification on these highly polar microcontaminats. Therefore, a novel highly sensitive method for speciation analysis of gadolinium is presented. For that purpose, the hyphenation of hydrophilic interaction liquid chromatography (HILIC) and inductively coupled plasma-mass spectrometry (ICP-MS) was employed. In order to enhance the detection power, sample introduction was carried out by ultrasonic nebulization. In combination with a novel HILIC method using a diol-based stationary phase, it was possible to achieve superior limits of detection for frequently applied gadolinium-based contrast agents below 20pmol/L. With this method, the contrast agents Gd-DTPA, Gd-DOTA and Gd-BT-DO3A were determined in concentrations up to 159pmol/L in samples from several waterworks in a densely populated region of Germany alongside the river Ruhr as well as from a waterworks near a catchment lake. Thereby, the direct impact of anthropogenic gadolinium species being present in the surface water on the amount of anthropogenic gadolinium in drinking water was shown. There was no evidence for the degradation of contrast agents, the release of Gd(3+) or the presence of further Gd species.