Applied methodologySpatial investigation of the elemental distribution in Wilson’s disease liver after d-penicillamine treatment by LA-ICP-MS
Introduction
Wilson’s disease (WD) is a rare autosomal-recessive inherited disease of the copper metabolism leading to a copper accumulation especially in the liver and the central nervous system [1]. WD is caused by the defective gene ATP7B, which encodes for a metal-transporting ATPase, responsible for the biliary excretion of excess copper [2], [3]. The diagnosis of WD is complex due to manifold hepatic and neuropsychiatric symptoms as well as high variability of laboratory results [4], [5].
A lifelong and continuous therapy is required for WD in order to maintain the copper homeostasis, allowing for a normal life expectancy in many WD patients [6]. Currently, copper chelating agents like d-penicillamine (DPA) and trientine, zinc salts, or a combination thereof are clinically applied for WD therapy [5]. Chelating agents are typically employed in order to remove excess copper from the organism and to cause a negative copper balance [5]. Zinc salts can be applied to induce metallothionein in the gastrointestinal tract and in hepatocytes, which shows a high affinity for copper due to a cysteine-rich structure [7], [8]. If a conventional therapy with chelating agents is not effective or a fulminant form of WD occurs, a liver transplantation becomes mandatory [9].
DPA treatment, which was first used in 1956, is the first-line therapy for WD [5], [10]. However, DPA may cause severe adverse effects and even worsening of neurological symptoms in WD patients [11]. Furthermore, upon discontinuation of the DPA treatment, a rapid clinical deterioration may take place, resulting in the necessity of a liver transplantation or even in the death of the patient [12]. This rapidly deteriorating liver status has been suggested to be the result of insufficient copper elimination by DPA in WD patient livers. In fact, massively elevated liver copper levels have been reported in WD patients despite decades of DPA therapy [13].
To investigate the distribution of remaining copper, spatially resolved techniques for elemental bioimaging such as laser ablation-inductively coupled plasma-mass spec-trometry (LA-ICP-MS) offer outstanding characteristics to detect copper within the liver tissue. LA-ICP-MS was first applied for elemental bioimaging in 1994 by Wang et al. and exhibits a high spatial resolution in a micrometer range as well as limits of detection in the μg·kg−1 range [14], [15], [16]. Additionally, analyte quantification is possible by internal and external calibration [17]. In the literature, different examples for elemental bioimaging in rat, sheep, and human liver tissues are described [18], [19], [20], [21], [22]. A recent study for the investigation of rat and human liver by LA-ICP-MS was published by Boaru et al. using a spatial resolution of 60 μm and an external calibration with matrix-matched standards made of mouse brain. That work focused especially on total elemental concentrations showing an age-dependent accumulation of copper, iron, and zinc in Atp7b deficient mice as well as an elevation of these metals in human WD liver. Although regions with elevated elemental concentrations within the rat and human liver samples were detected, no information on smaller structures within the liver tissue has been discussed due to the relatively low resolution of 60 μm in that study [21].
In the present study, LA-ICP-MS is used for high resolution elemental bioimaging of copper, iron, and zinc in livers of a DPA treated WD patient and a WD animal model, the LPP rat. The presented LA-ICP-MS method offers a spatial resolution of 10 μm spotsize and allows for quantification of physiological copper, iron, and zinc concentrations in liver tissue. Next to the determination of the total elemental concentrations in these rat liver samples, the small spotsize of 10 μm offers a spatial resolution sufficient for elucidation of a copper wash-out in proximity of blood vessels upon DPA therapy. A sample set including LPP rat liver samples with and without DPA treatment is analyzed. Atp7b−/− deficient LPP−/− rats with and without several weeks of DPA treatment are compared to unaffected LPP+/− controls. A zonal analysis is performed to depict the copper concentrations with respect to the distance to blood vessels in order to evaluate the copper concentration and distribution within the rat liver tissue. In addition to this, a human liver sample from a DPA treated WD patient has been analyzed by LA-ICP-MS.
Section snippets
Chemicals and reagents
All chemicals were used in the highest quality available. Copper (II) sulfate pentahydrate, iron (III) chloride, zinc (II) chloride, multi-elemental standard IV (1000 mg/L), nitric acid (65%, Suprapur), ethanol, and xylene were obtained from Merck KGaA (Darmstadt, Germany). Gallium standard solution (1000 mg/L) was purchased from SCP Science (Baie D´Urfé, Canada). Gelatine was obtained from Grüssing GmbH (Filsum, Germany). All solutions were prepared with doubly distilled water generated by an
Characteristics of the rat liver samples used for LA-ICP-MS analysis
Next to LA-ICP-MS analysis, our strategy involved standard protocols routinely used in the diagnosis of WD. The LPP−/− rats carry the WD-causing genotype Atp7b−/− and were either untreated or subjected to DPA treatment (Table 1). Unaffected healthy rats (LPP+/−, genotype Atp7b+/−) served as a control. Liver disease markers were highly elevated in untreated LPP−/− rats as compared to DPA treated LPP−/− rats or controls as reported before [23], [26]. Haematoxylin and eosin (HE) liver stains of
Conclusions
In this work, the suitability of LA‐ICP‐MS for the spatial investigation of the elemental distribution in liver samples of WD patients and a WD rat model is demonstrated. The applied LA‐ICP‐MS method features a spotsize of 10 μm suitable for the zonal analysis and appropriate limits of quantification for physiological element concentrations in control samples.
Results by LA‐ICP‐MS revealed copper hotspots distributed over the liver tissue within the diseased rat liver. The hotspots possibly
Conflict of interest statement
All authors declare that there are no conflicts of interest associated with this manuscript.
Acknowledgement
Parts of this study were supported by the Cells in Motion Cluster of Excellence (CiM – EXC 1003), Münster, Germany (project FF-2013-17).
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