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Mass Spectrometry Imaging, Inductively Coupled Plasma Mass Spectrometry Complementary bioimaging of Gadofluorine P in myocardial infarction in mice Rebecca Buchholz 1 , Fabian Lohöfer 2 , Michael Sperling 1,3 , Moritz Wildgruber 4 and Uwe Karst 1 1 Institute of Inorganic and Analytical Chemistry, University of Münster 2 Institute of Radiology, TU Munich 3 European Virtual Institute for Speciation Analysis (EVISA), Münster 4 Institute of Clinical Radiology, University Hospital Münster ■ Introduction Magnetic resonance imaging (MRI) is a widely used imaging technique in the daily clinical practice. To enhance the contrast during this examination, several different contrast agents are available. Because of their excellent paramagnetic properties due to five or seven unpaired electrons, Fe 3+ , Mn 2+ or Gd 3+ are mostly used. Since Gd 3+ is toxic in the free form, this probe is administered as a complex with aminocarboxylic acids. While the most gadolinium-based contrast agents (GBCAs) distribute systemically, some target specific GBCAs are under investigation as well. Gadofluorine P is one of these target specific contrast agent and shows high affinity towards the collagen-rich extracellular matrix (ECM), which is secreted in case of myocardial infarction (MI). The combination of two complementary bioimaging techniques is able to visualize this accumulation. Laser ablation with inductively coupled plasma mass spectrometry (LA-ICP-MS) is used to generate quantified images on an elemental level with high spatial resolution, while matrix- assisted laser desorption/ionization mass spectrometry (MALDI-MS) is used to validate the findings on a molecular level and provide further information regarding the distribution of for example phospholipids or heme b. ■ Materials and methods Animal study The animal study was performed in the research group of Prof. Moritz Wildgruber at the Institute of Clinical Radiology (University Hospital Muenster). Six weeks after an induced MI, the mice undergo MRI examination with injection of Gadofluorine P- solution as contrast agents. Afterwards the mice were sacrificed, the hearts were removed and snap frozen. Thin sections with a thickness of 10 μm were prepared with a cryomicrotome. Standard preparation For LA-ICP-MS analysis, matrix-matched standards based on gelatin were prepared for external calibration. Gelatin (10%w/w) was spiked with nine different concentrations ranging from 0 to 5000 μg/g Gd. Thin sections of the standards were also prepared with a thickness of 10 μm. Sample preparation For MALDI-MS imaging, the thin sections were placed on indium-tin oxide (ITO) slides. As a matrix is required, α-cyanohydroxycinnamic acid (CHCA) was deposited via sublimation onto the slide and the samples were afterwards recrystallized with 500 μl bidest. water and 50 μm methanol for 2.5 min. Analytical conditions For LA-ICP-MS analysis the ICPMS-2030 was coupled via a Tygon Ⓡ tubing to the laser ablation system LSX-213 G2+ (Teledyne CETAC) equipped with the HelEX II cell and a Nd-YAG laser with a wavelength of 213 nm. Helium was used as flush and transport gas for the ablation cell. The ICP-MS 2030 was equipped with nickel sampler and skimmer. No. SCA-116-005 Figure 1: Structure of Gadofluorine P.
Measurements were performed in collision gas mode with integration times of 100 ms for 31 P, 57 Fe, 66 Zn, 158 Gd and 160 Gd. For the calibration, 10 lines for each standard were analyzed with the same conditions as the sample (table 1). The MALDI-MS experiments were performed with the iMScope TRIO equipped with an ion trap – time of flight (IT-TOF) mass analyzer. Positive ion mode and a mass range from 700 to 1200 were selected. Additional experimental conditions can be found in table 2. The matrix was deposited with the iMLayer TM for 20 min. ■ Results LA-ICP-MS External calibration with matrix matched standards showed a linear correlation for a concentration range up to 5000 μg/g with a correlation coefficient R 2 of 0.997. For the used spotsize of 15 μm a limit of detection (LOD) of 43 ng/g Gd based on 158 Gd was determined and the limit of quantification (LOQ) based on the same isotope was 140 ng/g Gd (calculations based on Boumans [1]). Hematoxylin and eosin staining was performed to detect the area of myocardial infarct (marked with a black line) on a parallel thin section shown in figure 2. Figure 3 shows the microscopic images (a. and b.) of the two analysed thin sections. With LA-ICP-MS (c.), a homogenous distribution of the Gd in the healthy myocardium with an average concentration of about 50 μg/g was detected. The infarct region contains two times higher Gd concentrations of about 110 μg/g with maximum values up to 370 μg/g. Higher Gd concentrations can also be found in the ventricle due to the intravenous administration of the contrast agent. These distributions can be verified with MALDI-MS imaging (d.). In this experiment, only the protonated ligand of Gadofluorine P instead of the intact complex could be detected (e.). The main peak (m/z 1168.39) was used to create the image, which showed good correlation to the Gd distribution determined with LA-ICP-MS. Highest intensities of the molecular probe were found in MI and ventricle regions, whereas healthy myocardium shows low and homogenous intensities. SCA-116-005 Table 1: Experimental conditions used for LA-ICP-MS. Laser ablation LSX-213 G2 + Spot size 15 μm Scan speed 30 μm/s Shot frequency 20 Hz Cell gas flow 0.8 l/min ICP-MS ICPMS-2030 Plasma power 1.2 kW Sampling depth 5.0 mm Plasma gas 9.0 l/min Auxiliary gas 1.10 l/min Carrier gas 0.45 l/min Cell gas 6.0 ml/min Cell voltage -21 V MALDI iMScope TRIO Spot size 40 μm Sample voltage 3.5 kV Detector voltage 1.9 kV Accumulations 3 Laser shots 500 Laser frequency 1000 Hz Table 2: Experimental conditions used for MALDI-MS. Figure 2: H&E staining of mice heart tissue section.
■ Conclusion This application shows that the combination of an element selective (LA-ICP-MS) and a molecule selective (MALDI-MS) imaging technique is a powerful tool to visualize the distribution of target- specific gadolinium-based contrast agents in mouse heart tissue after myocardial infarction. High spatial resolution and quantification were achieved by means of LA-ICP-MS and the distribution was verified on a molecular level by means of MALDI-MS. © Shimadzu Europa GmbH 2018 www.shimadzu.eu Shimadzu Europa GmbH SCA-116-005 Figure 3: Microscopic images of the two parallel sections (a. and b.), quantified distribution of Gd determined with LA-ICP-MS (c.), distribution of the ligand from Gadofluorine P (d.) as well as the structure of the ligand and the theoretical spectrum (cyan bars) and the measured spectra (black line) with MALDI-MS (e.). iMLayer is a trademark of Shimadzu Corporation in Japan and/or other countries. Tygon is a registered trademark of Saint-Gobain Performance Plastics Corporation. ■ References [1] P.W.J.M. Boumans, Spectrochimica Acta 1991, 46 B, 641-665.
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