Accurate Measurement of Copper Overload in an Experimental Model of Wilson Disease by Laser Ablation Inductively Coupled Plasma Mass Spectrometry

Abstract

Wilson disease is a rare inherited autosomal recessive disorder. As a consequence of genetic alterations in the ATP7B gene, copper begins to accumulate in the body, particularly in the liver and brain. Affected persons are prone to develop liver cancer and severe psychiatric and neurological symptoms. Clinically, the development of corneal Kayser-Fleischer rings and low ceruloplasmin concentrations (<20 mg/dL) are indicative of Wilson disease. However, the detection of elevated hepatic copper content (>250 µg/g dry weight) alone is still considered as the best but not exclusive diagnostic test for Wilson disease. Presently, specific copper stains (e.g., rhodanine) or indirect staining for copper-associated proteins (e.g., orcein) are widely used to histochemically visualize hepatic copper deposits. However, these procedures only detect lysosomal copper, while cytosolic copper is not detectable. Similarly, elemental analysis in scanning electron microscope with energy dispersive X-ray analysis (EDX) often leads to false negative results and inconsistencies. Here, we tested the diagnostic potential of laser ablation inductively-coupled mass spectrometry (LA-ICP-MS) that allows quantitative analysis of multiple elements. Comparative studies were performed in wild type and the Atp7b null mouse model. We propose LA-ICP-MS as a versatile and powerful method for the accurate determination of hepatic copper in people with Wilson disease with high spatial resolution.

Analyzing the Therapeutic Efficacy of Bis-Choline-Tetrathiomolybdate in the Atp7b−/− Copper Overload Mouse Model

“…In recent years, we introduced novel LA-ICP-MS imaging protocols to identify and quantify changes in hepatic major, minor and trace metals in thin cryosections of Atp7b −/− mice [ 21 , 22 , 26 , 28 , 31 , 33 ]. Absolute concentration of various elements can be done by normalization to surrogate markers such as 13 C, indicating slice thickness and comparison to a standard, in which the concentration of individual elements are well-defined [ 36 ].…”

“…Previous studies have demonstrated hepatic Cu overload to be associated with many pathological changes at an ultrastructural level, including severe mitochondrial changes, increased numbers of peroxisomes, lipid droplet formation, occurrence of lipopolyosomes, and electron-dense lysosomal deposits [ 37 , 38 , 39 ]. We were able to demonstrate such electron-dense lysosomal deposits and fat droplets in liver specimens of Atp7b −/− mice using protocols recently established in our laboratory [ 31 ]. In most cases, these granular particles were arranged in lysosomal vesicles irregularly distributed within the tissue ( Figure S2 ).…”

“…We have previously shown that LA-ICP-MS imaging is well-suited to measure Cu overload in the Atp7b −/− mouse model ( Figure S1 ) [ 21 , 22 , 31 ]. In the present study, we used this methodology to evaluate the therapeutic efficacy of bc-TTM in this Wilson’s disease model.…”

“…Electron microscopic analysis of Atp7b −/− mouse liver tissue was done essentially as described before [ 31 ]. In brief, after dissection of the livers, small tissue samples with a size 1–2 mm were prepared and fixed in 3% glutaraldehyde prepared in 1× phosphate buffered saline (PBS).…”

“…In brief, after dissection of the livers, small tissue samples with a size 1–2 mm were prepared and fixed in 3% glutaraldehyde prepared in 1× phosphate buffered saline (PBS). Samples were washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), post-fixed in 1% Osmium tetroxide (OsO 4 ) (Roth, Karlsruhe, Germany) in 17% sucrose buffer (Merck), dehydrated through ascending ethanol series, and embedded in epoxy resin as reported before [ 31 ]. Ultrathin sections (70–100 nm) were prepared and spread on Cu/Rh grids (HR23 Maxtaform, Plano GmbH, Wetzlar, Germany).…”