TRACE METAL IMAGING IN DIAGNOSTIC OF HEPATIC METAL DISEASE Iuliana Susnea1 and Ralf Weiskirchen2* 1

Central Institute of Engineering, Electronics and Analytics (ZEA-3), Forschungszentrum Ju¨lich, D-52425, Ju¨lich, Germany 2 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, D-52074, Aachen, Germany Received 6 September 2014; revised 25 November 2014; accepted 2 December 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21454

The liver is the most central organ and the largest gland of the body that influences and controls a variety of metabolic and catabolic processes. It produces inconceivable many essential proteins, is responsible for the recovery of various food components, degrades toxins, mediates the bile production, and is involved in the excretion of unwanted metabolites. Several of these anabolic or catabolic functions of the liver depend on trace elements. These are either integral part of enzymes, cofactors, or act as chemical catalysts. Therefore, a lack of trace elements can lead to organ failure or systemic illness. Conversely, excessive hepatic trace element deposition resulting from genetic disorders, intoxication, extensive dietary supply, or long-term parenteral nutrition may cause hepatic inflammation, fibrosis, cirrhosis, and even hepatocellular carcinoma. Although specific serum parameters currently allow rough assessment of metal deficit and excess, the precise quantification of hepatic metal content in liver is presently only possible by different titration or staining techniques of biopsy specimens. Recently, novel innovative metal imaging techniques were developed that are on the way to replace these traditional methods. In the present review, we summarize the function of different trace elements in liver health and disease and discuss the present knowledge on how quantitative biometal imaging techniques such as synchrotron X-ray fluorescence microscopy, secondary ion mass spectrometry, and laser ablation inductively coupled plasma mass spectrometry enrich diagnostics in the detection and quantification of hepatic metal disorders. We will further discuss sample preparation, sensitivity, spatial resolution, specificity, quantification strategies, and potential future applications of metal bioimaging in experimental research and clinical daily routine. # 2015 Wiley Periodicals, Inc. Mass Spec Rev 34:1–21, 2015 Keywords: metal bioimaging; hemochromatosis; Wilson’s disease; liver; metal poisoning; synchrotron X-ray fluorescence; secondary ion mass spectrometry; laser ablation inductively coupled plasma mass spectrometry

Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract grant numbers: DFG, SFB, TRR57; Contract grant sponsor: Interdisciplinary Center of Biomedical Research.  Correspondence to: Prof. Dr. Ralf Weiskirchen, Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, D-52074 Aachen, Germany. E-mail: [email protected]

Mass Spectrometry Reviews, 2015, 9999, 1–21 # 2015 by Wiley Periodicals, Inc.

I. INTRODUCTION The liver plays a fundamental role in the metabolism of the body. It impacts energy supply and support, storage of nutritionals, synthesis of various proteins, hormones, lipids, sugars, and fats (Gressner & Weiskirchen, 2006). It metabolizes drugs and toxins, excretes unwanted excavated materials by acting as a gland that is capable to synthesize a complex mixture of bile liquids. Recent work has further demonstrated that individual liver cell subpopulations have important immunological functions by serving as antigen-presenting cells and forming major barriers that prevent the penetration of bacteria or viruses into the body or even attack these pathogenic organisms (Gressner & Weiskirchen, 2006). In all these hepatic activities metals play pivotal roles. They are either part of protein complexes, act as essential cofactors, or are indispensable catalysts in a plurality of biochemical reactions (Neuschwander-Tetri, 2007). Protein bound metal ions are critical for the function, structure and/or stability of these macromolecules. Moreover, catalytically active metal-containing proteins (i.e., metal enzymes) are frequently rate limiting factors in crucial reaction chains. Other metals are integral part of hormones that control important systemic effects (e.g., blood pressure) or convey stimulatory or inhibitory effects on responsive cells or target organs (Neuschwander-Tetri, 2007). As a consequence, the lack of an individual trace element can lead to shortcomings, failures, and illness. Vice versa, excess supply with an element that might be caused by genetic disorders, intoxication, exorbitant dietary intake (nutritional oversubstitution), organ failure, or during therapeutic treatments such as blood transfusion and long-term parenteral nutrition may result in elevated metal concentrations and deposits. In the liver metal overload results in intracellular reactive oxygen species (ROS) formation that induce necrosis and apoptosis of parenchymal cells (i.e., hepatocytes) that represent a major synthesis and storage sites of a multitude of cytokines (e.g., transforming growth factor-b, TGF-b) and chemokines (Gressner & Weiskirchen, 2006; Wasmuth & Weiskirchen, 2010). During cellular damage, these small, highly active biomolecules become released and provoke hepatic inflammation and activation of profibrogenic cells such as quiescent hepatic stellate cells (HSC) and portal myofibroblasts that are triggered to synthesize large quantities of extracellular matrix components such as collagens. When untreated, these changes induce a scar tissue formation (fibrosis), cirrhosis, and even hepatocellular carcinoma (Gressner & Weiskirchen, 2006).

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TABLE 1. Sources of hepatic metal imbalance Metal Fe

Mn

Cu Zn

Cd

Pb Hg As

Ag

Se

Mo Al

Tl Ni Cr Co Ba

Reasons for an abundance Genetic disorders causing excessive intestinal absorption (hemochromatosis, β -thalassemia; Aceruloplasminemia); transfusional Fe overload (repeated blood transfusion) Genetic disorders (SLC30A10 mutations); illness-induced Mn increase (cholestasis, hepatic failure); long-term parenteral nutrition; excessive Mn supplements; therapeutic usage of Mn oxidecontaining micro- and nanoparticles; occupational Mn exposure (manufacturers of batteries and ferro-alloy); air and road-side contamination (anti-knock agent in gasoline); usage of oral contrast agent in liver magnetic resonance imaging (e.g., CMC-001); suicide attempts (KMnO4, MnO2); insufficient biliary excretion; increased intestinal absorption during Fe deficiency Genetic disorders (Wilson disease, COMMD1 gene mutations); idiopathic Cu toxicosis; long-term exposure (drinking water, fungicides, pesticides, tobacco, metal industry)

Reasons for a lack Iron malabsorption; internal chronic and excessive menstrual bleeding (resulting in anaemia); nutritional deficiencies (strict vegetarian food consummation) High intake of Fe and Ca that inhibit proper intake of Mn; inadequate parenteral nutrition

Accumulation resulting from toxic environmental (industrial pollutant, alkaline batteries, pigments for paints or plastics, plating on metals): smoking; extensive dietary intake (e.g., extensive consummation of liver, mushroom, mussels, linseed, cacao, seaweed) Elevated Pb in drinking water, air (petrol additives, tobacco), and ceramic; occupational exposure; contamination in traditional remedies (Ayurveda); Pb accumulations in food (mussels) Dietary intake (Minamata disease); job-related inhalation (exposure to amalgam, leakage of Hg thermometers) Elevated uptake through contaminated drinking water, air and food; misuse as therapeutic agent; inhalation of As-containing substances (As2O3); suicide attempts; criminal poisoning Exposure to soluble Ag compounds or Ag dust (Argyria); high concentration of Ag in dental amalgam; exposure to Ag-containing antiparasitic drugs Intoxication with Se-containing drugs; inhalation or aliments (Selenosis)

not known

Suicide attempts; criminal poisoning; extensive contact with household rodenticides (Tl2SO4); TI exposure at smelters in the maintenance and cleaning of ducts and flues Elevated uptake through contaminated air; (harmless) increase in uptake during Fe deficiency, pregnancy and lactation Occupational exposure with Cr-containing compounds (e.g., metal abrasion) Inhalation or absorption during work in glass, enamel or ceramic factory or intake of Co-containing or contaminated substances Contamination of soluble Ba salts in contrast agent (BaSO4); intake by contaminated drinking water or inhalation

not known

Overmedication with Zn; insufficient dietary intake; malabsorption; long-term parenteral nutrition; genetic disorders (X-linked Menkes disease, ATP7A gene mutations) Overdoses of Zn in treatment of Wilson disease; zinc phosphide Insufficient dietary intake (extensive fasting, die poisoning; dietary intake of food stored for long-term in galvanised protein intake); extensive drinking of phosphate-containing containers); enduring exposure to Zn-containing fume (work in drinks (cola); continuing overdose with Ca; alcohol abuse; foundry) various inflammatory diseases affecting Zn uptake (Colitis ulcerosa); extensive blood loss; pathological sweating; insufficient complementation during pregnancy; increased Zn loss after usage of laxatives, cortisone and birth control pills

not known not known

not known

Monotonic alimentation (Vegans), long time parenteral nutrition; malabsorption; alcoholism; Bulimia nervosa; diabetes; various nephrotic syndromes and disorders (e.g., proteinuria); purge abusus; uncontrolled intake of cholesterol lowering drugs (statins); excessive menstrual bleeding or breast feeding Excessive dietary supplementation; inhalation/Intake of Mo- Congenital Mo cofactor deficiency; low soil concentrations in containing dust or soluble molybdates several geographical regions (China, Iran); non-Mo supplemented total parenteral nutrition; high blood levels of sulfite and urate Al phosphide poisoning; Al inhalation, ingestion and dermal not known absorption; uptake from acid foods that were stored in Al foil

The causes for hepatic imbalances with a specific metal can be very complex (Table 1). One frequent reason for elevated metal concentrations is the occurrence of genetic disorders such as hereditary hemochromatosis and Wilson’s disease (WD) that 2

not known

Insufficient dietary uptake (underfeeding, malnutrition); inadequate resorption in the gastrointestinal tract Insufficient dietary intake; long term intravenously feeding with non-Cr supplemented liquid diets Insufficient supply with Vitamin B12 not known

affect the uptake or clearance of a specific metal or groups of metals. Alternatively, excessively nutritional supply and intended or unintended metal exposure through the digestive or respiratory tract may result in systemic metal overload (Fig. 1). Mass Spectrometry Reviews DOI 10.1002/mas

METAL IMAGING IN LIVER DISEASE

While percutaneous absorption is normally prevented by the stratum corneum in healthy skin (Filipe et al., 2009), the exposure of metals to injured skin predicts intense metal influx. Likewise, long-term parenteral nutrition, blood transfusion, and the oral administration of metal-containing drugs are other potential possibilities that might affect endogenous metal balance (Poole et al., 2012). Similarly, internal metal intoxication might be induced in individuals suffering from any kind of kidney, gall, pancreas or bladder disease that affects secretion or clearance of elements. In all cases, chronic metal exposure results in elevated metal blood levels and accumulation in liver cells where they promote the formation of free radicals (ROS) that induce cell damage, inflammation, and aging (Pourahmad et al., 2003). Typical signs of liver damage are hepatocyte apoptosis and necrosis, Kupffer cell activation, and initiation of fibrogenic responses discernible in the activation of HSC and portal myofibroblasts outlined above. Therefore, it is obvious that excessive intake and accumulation of metals within the body affects liver function and health. Liver damage correlates with elevated serum activities of alanine transaminase (ALT) and aspartate transaminase (AST) and can be further identified by measurement of different serum metal binding proteins that directly or indirectly correlate with the concentration of individual metals (Kowdley et al., 2012). On the other hand, several specialized histopathological stains (e.g., Prussian blue for iron), chelometric assay, or chemical titration methods are routinely in use to determine and quantify metals in surgical liver specimen. All these methods allow metal

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quantification with different accuracy, reproducibility and expenditure. Traditionally, these methods are further combined with specific imaging techniques such as scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDX) that serve as confirmatory test in cases in which the non-invasive markers already suggest elevated hepatic metal concentrations (Hayashi et al., 2013). However, this approach is actually only appropriate to speciate, quantitate, or locate a limited spectrum of trace metals in the examined tissue. In supplement to these methods, several novel, innovative biometal imaging techniques were established during the last years. Some of these applications are already well established. In particular, synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become a significant add-on in biomedical research and are on the way to be introduced into daily routine clinical practice. One major advantage of these new techniques is that they allow multi-elemental and multi-isotopic metal measurement and quantification in one run thereby providing more comprehensive information. In the first part of this review, we will provide some clinical relevant diseases that are associated or caused by alterations in hepatic metal homeostasis. In the second part, we will describe the different biometal imaging techniques and give several concrete examples in which these techniques were already successfully applied in experimental liver research or in diagnostics of human liver failures.

FIGURE 1. Pathways of hepatic trace metal accumulation. Metals that are orally taken up are either absorbed in the stomach or bowel. After inhalation of metal-containing air, metals can be absorbed through the lungs and transferred to the blood stream. Unwanted dermal metal intake is rare and generally only possible in damaged skin regions. Poisoning with metals during clinical practice is possible during periods of long-term enteral, peripheral or total parenteral nutrition. Extensive medication/diets with metal-containing drugs/supplements and blood transfusion are other potential sources of metal poisoning. Likewise, internal intoxication with metals may result in patients suffering from kidney, gall, pancreas, or bladder failures resulting in insufficient metal clearance. Hepatic metal accumulation induces hepatic necrosis, Kupffer cell activation and proliferation of hepatic stellate cells and portal myofibroblasts.

Mass Spectrometry Reviews DOI 10.1002/mas

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II. METALS AND THE LIVER A. Iron in Liver Homeostasis and Liver Disease Iron (Fe) is the most abundant trace element within the body. It is integral part of a variety of different blood proteins (hemoglobin), muscles (myoglobin), and oxidative active enzymes (cytochromes, peroxidases, and catalases). Fe is needed only in small amounts as the body can rely on several mechanisms allowing recycling of this element. Nevertheless, Fe malabsorption, excessive internal or menstrual bleeding, and nutritional deficiencies that are caused by strict uptake of vegetarian food may result in anaemia (Harvey et al., 2005). Vice versa, genetic alterations, excessive Fe absorption, and faults during medication, exorbitant blood transfusion may provoke elevated hepatic Fe concentrations within the body (Berdoukas et al., 2013) (Fig. 2). Mutations within the Hereditary Hemochromatosis Gene (HFE) that induce HFE type 1 are the most frequent cause leading to hyperabsorption of dietary Fe and systemic Fe overload (Piperno, 2013). Under normal conditions, the HFE

protein physically interacts with the transferrin receptor 1 (TfR1) and impairs cellular uptake of transferrin-bounded Fe. The lack of Fe results in elevated synthesis and release of transferrin within the liver and HFE increases the intestinal release of Fe into the blood. When the HFE gene is mutated, the intestines perpetually interpret a strong transferrin signal as if the body is lacking Fe. As a consequence, the absorption of Fe from ingested foods is increased and Fe overload occurs (Pietrangelo, 2010). Hemojuvelin (HJV) acts as a positive regulator of hepcidin representing the master regulator of Fe homeostasis (Roetto et al., 2003). Mutations within this gene are responsible for the vast majority of juvenile type 2A hemochromatosis patients (Papanikolaou et al., 2004). Therefore, biological alteration in HJV function results in low hepcidin levels leading to increased intestinal Fe absorption. Hepcidin itself inhibits Fe transport across the gut mucosa, thereby preventing excess Fe absorption and maintaining Fe homeostasis within the body. Likewise, mutations in the HAMP gene encoding Hepcidin may be associated with increased Fe absorption and provoke severe, juvenile hemochromatosis type 2B (Rosetto et al., 2003).

FIGURE 2. Pathogenesis of iron overload disease. Excessive Fe deposits might result from various mutations of genes that are involved in the control of Fe homeostasis. The most frequent are genetic disorders that give rise to different types of Hereditary Hemochromatosis affecting the genes HFE (Hereditary Hemochromatosis), HJV (Hemojuvelin), HAMP (Hepcidin Antimicrobial Peptide), TFR2 (Transferrin Receptor 2), and SLC40A1 (Solute Carrier Family 40 member 1, Ferroportin 1) that together control body Fe homeostasis. Other causes of Fe overload are neonatal hemochromatosis, acquired hemochromatosis, various types of thalassemia, or mutations within the CP gene encoding Cerulopasmin (also known as Ferroxidase). Elevated concentrations of free intracellular Fe induce formation of reactive oxygen species (ROS), mitochondria dysfunction, protein and membrane impairment, DNA damage, and alterations within lipid metabolism. Necrotic hepatocytes release large quantities of pro-fibrogenic cytokines (e.g., TGF-b1) that in combination with ROS induce inflammation and organ fibrosis. Untreated liver damage further results in cirrhosis or even hepatocellular carcinoma.

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Another important gene involved in the control of Fe homeostasis is the Transferrin Receptor 2 that mediates cellular uptake of transferrin-bound Fe. Mutations within this gene have been linked with hereditary hemochromatosis type III (Camaschella et al., 2000). Another group of mutations that potentially result in Fe overload are those in which hemoglobin is unduly degraded, insufficiently formed, or produced in altered forms. These diseases affect a large variety of genes that are grouped within the globin gene clusters located on human chromosomes 11 and 16 (Muncie & Campbell, 2009). The various thalassemia variants are named after the globins, which are not formed in sufficient quantities: a-and b-thalassemia. Most mutations are inherited autosomal recessive disorders that occur mainly in the former malaria areas in the Mediterranean (e.g., Malta, Sardinia, Sicily, Greece, Cyprus), the Near East, and in the population of African descent. Commonly, the diagnosis of thalassemias is made on the basis of typical clinical correlates, and secured in Hemoglobin electrophoresis and by genetic testing. The deposition of hepatic Fe (hemosiderosis) during thalassemias is diagnosed either by a liver biopsy with subsequent Prussian blue staining (Saxena, 2010), by Fe superconducting quantum interference device (SQUID), or special forms of magnetic resonance imaging (MRI) (Nielsen et al., 1995; Berdoukas et al., 2013). Symptomatic treatment is often only possible with regular blood transfusions that may further exacerbate hepatic Fe deposit. Aceruloplasminemia is a rare inherited metabolic disorder that is characterized by a complete absence of the ferroxidase activity of Caeruloplasmins. This enzyme acts as a copper storage and helps to transport Fe out of the cells. Hereditary deficiency of this enzyme leads to abnormal accumulation of Fe in reticuloendothelial cells and hepatocytes (Mukhopadhyay, Attieh, & Fox, 1998). Regardless of the cause, elevated Fe concentrations are most often associated with red blood cell break down and focal or general deposition of Fe storage complexes. This phenomenon is called hemosiderosis and primarily occurs in the hepatocytes. Connected thereto is the intracellular oxidation of organic substrates in the Fenton reaction that is a primary source of intracellular ROS formation (Crichton et al., 2002). This results in oxidative stress, protein and membrane damage, mitochondria dysfunction, DNA breakage, and in synthesis and release of inflammatory profibrogenic cytokines such as TGF-b that drives fibrogenesis (Fig. 2). eside these genetic disorders, there are a large number of infection diseases that also impact hepatic metal homeostasis. In the life cycle of the Malaria parasite Plasmodium for example, the merozoites enter into the red blood cells of the host and degrade hemoglobin. The protozoa detoxifies heme by converting it into a redox-inactive Fe(III) polymer called hemozoin (Pandey & Tekwani, 1996). This characteristic “malaria pigment” is then released in high quantities and found in many tissues including liver. Similarly, several other blood-feeding parasites (Rhodnius, Schistosoma) produce similar insoluble disposal Fe-enriched products.

B. Copper Metabolism and Liver Disease Copper (Cu) is an essential cofactor in the respiratory chain and an ingredient of a variety of diverse proteins and metalloenzymes Mass Spectrometry Reviews DOI 10.1002/mas

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involved in the maintenance of diverse biological functions including growth control, development, red blood cell formation, cholesterol and glucose metabolism, as well as immune regulation. Cu is primarily absorbed in the gut and transferred to the liver in an albumin-bound form (Adelstein & Vallee, 1961). The uptake into the enterocyte is mediated through the highaffinity Cu transporter 1 (Ctr1) (Lee, Prohaska, & Thiele, 2001). Intracellular transfer of cytosolic Cu to the transmembrane ATPase A protein that is located in the trans-Golgi network is mediated by carrier proteins known as Cu chaperones (Antioxidant protein 1, ATOX1; Metallothionin, MT) (Fig. 3). From this compartment Cu is incorporated by the Cu-transporting ATPase 1 (ATP7A) into Ceruloplasmin exhibiting that catalyzes the oxidation of Fe2þ into Fe3þ. Alternatively, in conditions of Cu saturation, the ATP7A protein directly eliminates excess Cu from the cell into the portal vein from which it is taken up by liver cells in a similar way. Linking of Cu to Ceruloplasmin, release into the blood, and elimination through bile are mediated within the liver by ATP7B, a Cu-translocating P-type ATPase. This excretion process is further regulated by several other ATP7B binding proteins that endogenously interact with this Cu regulator, thereby influencing key cellular signalling cascades (Coronado et al., 2005; de Bie et al., 2006). Based on this complex network of proteins involved in control of Cu uptake, transport, distribution and excretion it is not surprising that Cu homeostasis might be altered by several mutations affecting any of the above mentioned genes (Rosencrantz & Schilsky, 2011). Both, the lack and excess in Cu can lead to tissue injury and disease. Wilson’s disease (WD) is an inherited autosomal recessive genetic disorder in which mutations within the ATP7B gene occur. Patients affected by these mutations show severe hepatic Cu accumulations and exhibit various neurological disturbances (Huster, 2010; Johncilla & Mitchell, 2011; Rosencrantz & Schilsky, 2011). However, the histopathologic spectrum of Cuinduced toxicosis is extremely variable (Huster, 2010; Johncilla & Mitchell, 2011; Rosencrantz & Schilsky, 2011). Experimental studies in ATP7b deficient mice suggest that elevated Cu concentrations induce oxidative stress along with severe dysfunction of mitochondrial energy production resulting in molecular impairments that are causally involved in the formation of the liver and neurological abnormalities (Sauer et al., 2011). In humans, the occurrence of Cu deposit in the cornea causes the typical Kayser–Fleischer rings. Actually, the measurement of biochemical parameters such as urine Cu and serum ceruloplasmin, genetic testing, intravenous radiocopper loading or D-penicillamine challenge tests, ultrasound scan of the liver, and magnetic resonance imaging of the brain are presently important diagnostic strategies in WD diagnosis (Huster, 2010; Rosencrantz & Schilsky, 2011). However, all these options are sometimes unsuitable to rule out WD and several patients that do not possess the characteristic clinical symptoms are detected too late and develop hepatic or cerebral damage (Bennett & Hahn, 2011). Therefore, earlier recognition of the disease is still a challenging demand. Currently, novel technologies in Cu imaging including nearinfrared fluorescent sensors (Hirayama et al., 2012) and quantitative positron emission tomography of Cu metabolism (Peng et al., 2012) are experimentally investigated. Comparable, several mutations within the related ATP7A gene are identified that impair proper intestinal Cu 5

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FIGURE 3. Pathogenesis of Wilson disease. Free Cu (indicated as green circles) enters the body through the digestive tract. Specific Cu transporters (Ctr) carry Cu inside the cells, where it is bound to metallothionein (MT) or the antioxidant protein 1 (ATOX1) transporting it to the trans-Golgi network. When saturated, a plasma membrane Cu-transporting ATPase (ATP7A) that is normally localized predominantly in the trans-Golgi network is relocalized to the plasma membrane where it triggers Cu efflux. Cu homeostasis is further controlled by ATP7B that links Cu to Ceruloplasmin which has eight specific binding sites and releases Cu into the bloodstream or alternatively secretes it into the bile. Mutations within the ATP7B gene are causative for WD in which chronic excess Cu concentrations trigger hepatic inflammation, fatty liver, fibrosis and cirrhosis.

absorption or prevent shuttling from the Golgi apparatus to the cell membrane. Respective patients carrying these mutations suffer from Menkes disease and have an unbalanced Cu distribution with Cu accumulation in some tissues, while others have severe lack and show infant growth failure and deterioration of the nervous system. Since the ATP7A gene is located on the X chromosome, the prevalence of Menkes disease is more frequently observed in males (Menkes et al., 1962). In regard to the liver, it is noteworthy that beside these lossof-function mutations, there are some rare cases of patients with idiopathic Cu toxicosis suggesting that several other genes influencing hepatic Cu homeostasis are still unidentified. For example, one 11-year-old patient without any ATP7B mutation was identified presenting increased urinary Cu excretion without other typical signs of Cu overload (Kayser–Fleischer rings, neurologic alterations), while the liver specimen showed active hepatitis and cirrhosis (Hayashi et al., 2012).

C. Zinc and Liver Biology This trace element is required for a broad range of biological activities and acts as an essential cofactor for several hundred enzymes and zinc (Zn) finger-containing transcription factors (Prasad, 2013). Overdosing occurs only very rarely when Zn is given in very high concentrations (Berholf, 1988). Animal 6

experimentation has further shown a significant protective effect of Zn in the regulation of antioxidant status and in ameliorating the altered hepatic histoarchitecture in nickel-, lead-, and lithium-intoxicated animals (Bandhu et al., 2002; Sidhu et al., 2004; Chadha, Bhalla, & Dhawan, 2008). Zn homeostasis is controlled by several hormones and second messengers including glucocorticoids, glucagon, cAMP, and epinephrine. The liver mediates uptake from blood carriers (albumin) and distribution within the body (Cousins, 1986). Impaired Zn absorption has been already well documented in patients suffering from alcoholic and non-alcoholic liver disease (Sullivan, Jetton, & Burch, 1979; Karayalcin, Arcasoy, & Uzunalimoglu, 1988). Acutely toxic intake and excessive pharmaceutical uptake of Zn during prolonged periods of supplementation (e.g., in treatment of WD) or chronically uptake of relatively large amounts (e.g., intake of food stored for long-term in galvanized containers) may result in severe Cu deficiency associated with typical symptoms of hypocupremia, anemia, leucopenia, and neutropenia (Fosmire, 1990). In addition, alterations in immune response, white blood cell function, and in lipoprotein blood profiles were reported (Chandra, 1984). Research conducted on animals as well as on human subjects has shown that Zn deficiency is an important factor in the development and progression of malignancy, while appropriate supplementation is efficacious in the prevention and treatment of several cancers Mass Spectrometry Reviews DOI 10.1002/mas

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(for review see Dhawan & Chadha, 2010). In a small cohort of hemodialysis patients, a progressive increase in low-density lipoprotein (LDL) cholesterol concentration was observed after Zn-supplementation for 90 days, suggesting that Zn improves serum levels of cholesterol in these patients (Chevalier et al., 2002). Zn supplementation is therefore widely propagated and unwanted overdosage in principle possible. In particular, it was shown that extensive dietary Zn supplementation increases the expression of metallothionein impairing normal Cu uptake (Sullivan, Burnett, & Cousins, 1998).

D. Hepatic Manganese Content In liver, manganese (Mn) is an important trace element for a number of enzymes including arginase 1 that catalyzes the last step in the hepatic urea cycle in which arginine is cleaved to ornithine and urea. In animals, the lack of Mn can result in impaired insulin production, alterations in lipoprotein metabolism, impaired oxidant defense system, and perturbations in growth factor metabolism (for review see Keen et al., 1999). When rats are treated orally with high doses of Mn salts, retention of excess concentrations of Mn are not detectable after 14 days in any tissue suggesting that the rate of accumulation and overall toxicity of Mn is rather low (Holbrook et al., 1975). In line with that assumption, confirmed cases of Mn toxicosis in humans are currently restricted to cases of exposure to high levels of airborne Mn, and to cases in which Mn excretory pathways are malfunctioning (Keen et al., 1999).

E. Selenium and Selenoproteins in Liver Health Although selenium (Se) is a trace element nutrient that functions as cofactor in several antioxidant enzymes such as glutathione peroxidases, thioredoxin reductase, and selenoprotein P, no specific deficiency conditions have been reported in humans so far (Alexander, 2007). Interestingly, the complete family of selenoproteins that contains 25 members (i.e., the selenoproteome) in humans is the only known class of proteins for which expression is determined by the presence of a special tRNA (Carlson et al., 2004). Liver-specific disruption of the respective tRNA and complete deficiency of selenoproteins causes liver necrosis and death (Carlson et al., 2004). Supplementation with Se effectively reduces the severity of Zn deficiency in diabetic rats (Fatmi et al., 2013). However, the indiscriminate use of Se supplements that in human is supposed to protect against oxidative stress and type 2 diabetes cannot be justified (Rayman & Stranges, 2013).

F. Cobalt and Cobalt-Containing Vitamins Cobalt (Co) is integrated into the catalytic center of Vitamin B12. This vitamin is synthesized by gut bacteria and absorbed by the intrinsic factor-mediated gastrointestinal system (Watanabe et al., 2013). Vitamin B12 is involved in the form of its two coenzymes (i.e., methylcobalamin and 5-desoxyadenosylcobalamin) in various metabolic reactions. It has important cofactor functions in the field of protein metabolism, functioning of the brain and nervous system, and in red blood cell formation. Furthermore, it contributes to the regeneration of the mucous membranes and generally supports cell growth and cell division. In the degradation of fatty acids and branched chain amino acids Mass Spectrometry Reviews DOI 10.1002/mas

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odd 5-desoxyadenosylcobalamin catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. As most of the vitamin is stored in liver, hepatic alterations associated with reduced hepatic clearance result in elevated plasma concentrations of cobalamin, corrinoids and their analogs (Lambert et al., 1997).

G. Aluminum, a Potential Risk Factor for Liver Disease Aluminum (Al) is the most widely used non-ferrous metal in daily life. Although it is a remarkably non-toxic element (Llobet et al., 1987; Nordberg & Nordberg, 2009), environmental release, its high abundance, medical exposure, excessive dietary intake, contaminated drinking water are factors that may cause unwanted high intake of Al (Becaria et al., 2006). There are a wide variety of human diseases which have been linked to Al exposure including diseases of the bones, muscles, gastrointestinal tract, and brain (Exley, 2009). Owing to the hard acid properties of this metal and its pro-oxidant capabilities, this trivalent metal readily perturbs Fe homeostasis, disrupts biological membranes, enhances ROS formation, damages DNA, thereby promoting mitochrondrial dysfunction and intracellular lipid accumulation in hepatocytes (Mailloux, Lemire, & Appanna, 2007; Mailloux & Appanna, 2007; Kumar, Bal, & Gill, 2009; Peto, 2010). In addition, acute intoxication with Alphosphide, a widely use insecticide and rodenticide, leads worldwide to high mortality rates (Anand, Binukumar, & Gill, 2011). Al has no known biological function (Becaria, Campbell, & Bondy, 2002) and sources of medical intoxication have been identified (Exley, 2009). Therefore, the medical intoxication with Al is nowadays rare. However, Al contamination is still a problem (Jaffe, Liftman, & Glickman, 2005). Mitochondrial metabolism and aerobic respiration are central to the physiological function of hepatocytes and mitochondria are the main site of the biological action of Al (Mailloux, Lemire, & Appanna, 2011). In line with this assumption, the exposure of human hepatoma cells to Al contributes to a hypoxic environment that promotes oxidative stress and stimulates the anaerobic metabolism (Mailloux, Lemire, & Appanna, 2011). In addition, excessive uptake of Al salts (i.e., Al-sulphate) impacts Fe deposition, transferrin receptor expression, accelerates features of senescence in adult mice liver, and stimulates the deposition of collagen and laminin (Stacchiotti et al., 2008). Based on all these observation, it is probable that prolonged Al intake or exposure is associated with disease formation in many organs. Noteworthy, the liver is a likely sink for systemic Al and as such biliary excretion must be considered a potentially significant pathway for the excretion of systemic Al defects in bile production or excretion (Exley, 2009). Patients suffering from liver disease are therefore at greater risk for Al accumulation.

H. Cadmium and Hepatic Tumorigenesis Cadmium (Cd) is a toxic transition metal of considerable environmental and occupational concern that has carcinogenic potential for lung, liver, pancreas, and stomach (Waalkes, 2003). This heavy metal accumulates primarily in the liver and kidney where it is biological sequestered by binding to Metallothioneins (MT) in a similar manner as Zn (Klaassen, Liu, & Choudhuri, 1999). Therefore, if humans lack MT or have alterations in Zn metabolism, the normally tolerated intracellular Cd concentration might become toxic. In a cross-sectional study it was shown 7

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that environmental Cd exposures are primarily associated with an elevation in serum liver enzyme levels in humans (Kang et al., 2013). Also persons that are occupationally exposed to Cd by working in a primary metal or battery producing industry might be at risk for Cd poisoning. The classical symptoms of Cd poisoning such as softening of the bones and kidney failure are paradigmatic documented in the “Itai-Itai” disease that was caused in the past by mass Cd poisoning due to mining in the Toyama Prefecture located in the Hokuriku region of Japan (Nordberg, 2009). Likewise, extensive consumption of tobacco products might be another potential source for elevated Cd intake (Waalkes, 2003). In acute Cd overload, elevated levels of ROS are generated resulting in tissue damage (Liu et al., 2011). In contrast, it was shown that during adaptation to chronic Cd exposure ROS production is reduced in rat liver cells (Qu et al., 2005). However, in respective cells the chronic exposure to cadmium triggers oncogene overexpression and alters the activity of critical transcription factors such as AP-1 and NF-kB that are linked to tumorigenesis and malignant cell transformation (Qu et al., 2005). A recent study has further shown that Cd exhibits direct cytotoxic effects in diverse human and rat liver cell lines (Saı¨di et al., 2013). These findings strongly suggest that particular culminating concentrations and chronic Cd exposure are liver toxic and should be detected as early as possible.

I. Chromium and Glucose Metabolisms Chromium (Cr) III occurs in trace amounts in foods and water and has genotoxic and mutagenic potential (Stearns, 2000). Although there is no indication that the shortage in Cr results in defects in humans, some historical reports argued that Cr is an essential factor that maintains normal glucose tolerance in rats (Schwarz & Mertz, 1959). Based on these findings, it was supposed that this element increases the action of insulin and that a special chemical form, termed Glucose Tolerance Factor, a biologically active complex of Cr and nicotinic acid is suitable for oral medication for diabetes (Weksler-Zangen et al., 2012). In contrast to this assumption, a recent meta-analysis indicates that in patients with type 2 diabetes mellitus, Cr supplementation has no significant effect on body mass index, lipid profiles, and glycosylated hemoglobin (i.e., HbA1c) that is a diagnostic indicator of the average blood glucose level over the last eight weeks (Abdollahi et al., 2013). However, risk analysis in a Chinese cohort showed that occupational Cr poisoning led to an increased risk of lung or liver cancer in male workers (Yang et al., 2013). Again, these results demonstrate that early detection of poisoning is potentially essential to prevent metalinduced organ impairment.

J. Molybdenum and Molybdenum-Containing Enzymes Molybdenium (Mo) is an essential micronutrient which is part of some Mo-containing enzymes that generally catalyze the transfer of an oxygen atom to or from a substrate. In respective enzymes (e.g., sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase), Mo is complexed in a special arranged scaffold termed Molybdenum cofactor that causes a change in the oxidation state during reaction (Mendel, 2013). In patients lacking this essential cofactor the activity of these enzymes is 8

decreased and respective persons have severe neurological abnormalities, dislocated ocular lenses, and mental retardation (Johnson et al., 1980). In addition, urinary excretion of several sulfur-containing compounds is increased, while sulfate and urate levels are drastically reduced (Johnson et al., 1980). Excessive feeding with high dietary Mo containing chow in rabbits revealed that this element primarily accumulates in kidney and liver and is associated with generation of free radicals and increased creatine kinase activity (Berse´nyi et al., 2008). Presently, there is no example reported in which Mo deficiency or excess was shown to cause hepatic lesions in humans. However, there is on the other hand clear evidence that diets enriched in tetrathiomolybdate that acts as Cu chelator promote a decline in hepatic Cu and ceruloplasmin activity and induce clinical signs of Cu deficiency (Mills, El-Gallad, & Bremner, 1981). Therefore, thiomolybdate complexes are still attractive drugs for the treatment of WD (Medici & Sturniolo, 2008). The mutual influence from Mo and Cu further suggests that simultaneous imaging and quantification of these metals might be diagnostically meaningful.

K. Lead and Lead Poisoning Lead (Pb) and Pb compounds can be absorbed through food, by inhalation or through the skin. There is a significant difference whether Pb-based poisoning is induced by inorganic or organic compounds. Inorganic Pb poisoning, also known as saturnism or plumbism, damages the central and the peripheral nervous system. It further affects blood formation, induces gastrointestinal disorders and kidney damage. Absorbed or inhaled inorganic Pb is not metabolized and excreted unchanged, primarily in the urine. Alternative pathways of excretion may include secretion into the sweat, bile, gastric fluid, and saliva (Rabinowitz, Wetherill, & Kopple, 1976). Once absorbed, Pb is first bound to erythrocytes and is dispersed into liver, kidney, lung, and other soft tissues (Rabinowitz, Wetherill, & Kopple, 1976). Most of the body burden of Pb is found in the bone and the observed concentrations there greatly exceed those located in soft tissues (Barry, 1975). Most often, in male soft tissues Pb concentrations are found to be 30% higher than in equivalent female tissues (Barry, 1975). Organic Pb that may be taken up from the environment (e.g., tetraethyl- and tetramethyl-Pb in leaded gasoline) undergoes oxidative dealkylation within the liver by a cytochrome P450-dependent monooxygenease reaction (for review see Patrick, 2006). At non-toxic concentrations, Pb (like Hg) was noted to aggravate oxidative stress toxicity suggesting that small traces of this element represent already a risk factor that is noteworthy to be detected at the earliest possible point (Saı¨di et al., 2013).

L. Vanadium and its Toxicity Acute poisoning with vanadium (Va) in workers can manifest itself in a number of symptoms but genetic and related effects in humans chronically exposed to vanadium compounds are actually controversially discussed (Altamirano-Lozano et al., 2013). Studies in mice have shown that Va-pentoxide inhalation already for 1 hr twice a week for 5 weeks resulted in elevated aminotransferase activities, higher lipid peroxidation, oxidative stress, inflammatory infiltration, binucleation, and meganucleus formation in the liver (Cano-Gutie´rrez et al., 2012). These Mass Spectrometry Reviews DOI 10.1002/mas

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alterations indicate that exposure to Va is rather toxic and recommend that elevated hepatic Va concentrations should be recognized instantaneously.

M. Nickel and Nickel Intake Exposure to nickel (Ni) occurs by breathing air, drinking contaminating water, consuming Ni-loaded food, or by extensive smoking tobacco containing large quantities of Ni. Long term occupational contact to Ni appears also in persons working in electroplating industries, or factories producing alloy, stainless steel, Ni-Cd batteries, or recycling metal compounds. The uptake of Ni is possible by inhalation, dermal contact, and gastrointestinal ingestion. It is known that Ni induces generation of ROS and increased lipid peroxidation in various metabolically active tissues (Das & Buchner, 2007). The primary target organs for Ni accumulation are the kidneys and the lungs but other organs including liver, spleen, heart and testes may also be affected to a lesser extent (Cameron, Buchner, & Tchounwou, 2011). In human hepatoma cells, the exposure to Ni2þ resulted in a significant PI3K-dependent activation of AKT/PKB (Eckers, Reimann, & Klotz, 2009), that represents one critical mediator in the pathogenesis of hepatocellular carcinoma (Wang et al., 2013).

N. Silver, Gold, and Mercury Accumulation Silver (Ag), Gold (Au), and Mercury (Hg) have no known biological functions in humans. However, Ag ions are biologically active. They readily interact with proteins, amino acid residues, free anions and all kind of receptors on cell membranes (Lansdown, 2006). Improper exposure to elemental Ag, Ag dust or Ag-containing compounds that are included for example in most photographic materials may lead to Ag accumulation and deposition in the skin (Argyria), eye (Argyrosis) or other organs. Ag products are nowadays broadly used for medical indications (Fung & Bowen, 1996; Lansdown, 2006). In addition, Ag nanoparticles with variable sizes are exploited and applied in electronics, bio-sensing, clothing, food industry, paints, sunscreens, cosmetics and diverse medical devices without precise knowledge on potential side effects on human health (Ahamed, Alsalhi, & Siddiqui, 2010). In nanotechnology also Au nanoparticles are frequently employed. Nanoparticles of yellow Au melt at much lower temperature than those made out of Ag and are not cytotoxic even at high concentrations. However, they accumulate in cells that have strong phagocytic capacity (Villiers et al., 2010). Hg is a highly toxic element that is used in thermometers, barometers, manometers, fluorescent lamps and in many other daily life and medical devices (e.g., cosmetics, amalgam). Hg in the form of one of its common ores, cinnabar (HgS), is applied in various traditional medicines in Asia and for art in ancient South America. However, consumed in overdose or chronically incorporated even at therapeutic doses, these medicines evolve strong adverse effects. In a large U.S. cohort, it was demonstrated that the averaged mean concentration of inorganic Hg increases significantly during aging and is related with several biomarkers indicating damage in liver, immune system, and pituitary gland (Laks, 2009). Intoxication with bivalent Hg was further shown to decrease total cytochrome P450 content, hepatic microsomal protein quantities, and CYP2E1 activity in rats suggesting that exposure Mass Spectrometry Reviews DOI 10.1002/mas

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to Hg potentially generates free radicals (Alexidis, Rekka, & Kourounakis, 1994). Hg can be absorbed through the skin and mucous membranes and Hg vapors can be inhaled. A recent study that was performed in the saltwater fish Goliath grouper (i.e., Epinephelus itajara) revealed that total Hg concentration was greatest in liver tissue, followed by kidney, muscle, gonad, and brain (Adams & Sonne, 2013). In mice it was shown that organ specific Hg accumulation is dependent on gender and genetic factors suggesting that a large variation in Hg toxicokinetics exists in rodents and potentially in humans (Ekstrand et al., 2010).

O. Arsenic, Thallium, and Barium Arsenic (As) and As compounds are potent poisons that uncouple oxidative phosphorylation and disrupt ATP production. Trivalent As oxides have high affinity for thiols that are present within cysteine residues and in cofactors (e.g., lipoic acid, coenzyme A) of many essential enzymes. Poisoning with As may occur in persons working in industries that use inorganic As in wood preservation or production of glass, nonferrous metal alloys, and electronic semiconductors. As is well absorbed from the gastrointestinal tract and the induction of malignant transformation and hepatocellular adenoma and carcinoma is well documented in mice, rats, and humans (Liu & Waalkes, 2008). Likewise, the nonessential heavy metal thallium (Tl) is extremely toxic. Monovalent Tl compounds have a high aqueous solubility, are nearly tasteless, and most often colorless. Therefore, these substances are ideal ingredients for rat poisons that give rise to frequent intoxications caused by accident or criminal intent. Tl can accumulate in various cells, have weak mutagenic properties, affect cell-cycle progression, interfere with the metabolism of other metal cations (e.g., potassium) thereby mimicking or inhibiting their action (Rodrı´guezMercado & Altamirano-Lozano, 2013). Barium (Ba) is used in the manufacturing industry and in medicine as radiopaque material. Adverse health effects may occur due to absorption of soluble Ba compounds (chloride, nitrate, and hydroxide) that are toxic to humans. Autopsy in patients who died from fatal Ba-chloride poisoning showed subendocardial hemorrhage, visceral petechiae, and fatty changes in the liver (Ananda, Shaohua, & Liang, 2013). However, studies in rats have shown that Ba does not accumulate in the liver suggesting that the effects on fat metabolism in liver might occur indirectly (Edel et al., 1991).

P. General Remarks on Biometal Imaging Techniques Presently, element analysis and estimation of metal content in liver biopsy is often directly measured by titration, specific stains (e.g., Prussian blue) or alternatively more elegantly determined in transmission electron microscope (TEM) with electron energy loss spectroscopy (EELS) and in scanning electron microscope (SEM) with energy dispersive x-ray analysis (EDX) (Jonas et al., 2001). SEM-EDX for example is used for chemical analysis and determination of the elemental composition of microscopic particles or regions within a sample by in situ measuring the energy and intensity distribution of X-ray signals generated by a focused electron beam on the specimen (i.e., biopsy) (Fig. 4A). It relies on the investigation of 9

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an interaction of some source of X-ray excitation and the sample. This results in a unique set of peaks on the X-ray spectrum that is characteristic for each element. In brief, the necessary equipment for this technique is based on an excitation source that transmits an electron beam, an X-ray detector that converts X-ray energy into voltage signals, a pulse processor and an analyzer that processes the signals and matches them to individual atoms. Based on the fact that this technique allows discriminating of metals, it is used to corroborate or even score metal disorders in organ tissue. However, the accuracy of this technique is somewhat limited. Firstly, the analyst who performs the analysis has a bias and restricts his analysis to material deposits that are visually detectable. Secondly, several elements have overlapping peaks in EDX that under certain constellations prevent the precise assignment of signals to element. Thirdly, quantification of individual element concentration is impossible or only hardly to achieve. Finally, the accuracy of the spectrum can also be affected by the nature of the sample per se, i.e., the matrix, in that way that inhomogeneous samples result in inadequate or dissimilar excitations. Various direct histopathology stains of metals are applied by pathologists to detect the presence of a specific

metal deposit in biopsy specimens. In Prussian blue stain, Fe3þ that is not bound to heme forms an insoluble blue precipitate when incubated with potassium hexacyanoferrate (II) in hydrochloric acid. Although this allows identifying Fe deposits and measuring Fe semi-quantitatively within the analyzed tissue, the reaction is influenced by many factors including pH, exposure time, and storage temperature. Moreover, other metals (e.g., cesium) have affinity for ferric hexacyanoferrate and interfere with precise quantification (Faustino et al., 2008). Because of all these limitations, there is an urgent diagnostic need for novel metal imaging techniques that allow precise and simultaneous quantification of various trace metals in tissue. In biomedical research, synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have developed dramatically. All three methods allow accurate measurement of metals with high spatial resolution. The main characteristics of these and other metal imaging techniques are compared in Table 2. In the following sections we will discuss the potential application of SXRF, SIMS, and LA-ICP-MS in diagnostics.

FIGURE 4. Principles of EDX-SEM, SXRF, and SIMS. (A) EDX-SEM is an analytical technique that relies on the interaction of X-ray excitation and a sample. Therefore, a high-energy beam of charged particles (electrons or protons) or a beam of X-rays is focused into the sample and the number and energy of the X-rays emitted from respective specimens are measured by an energy-dispersive spectrometer. By this technique the different metals are recognized by their different element-specific energy spectra. (B) In Synchrotron X-ray Fluorescence (SXRF), the specimen is raster-scanned by focussed X-rays that pass through the specimen. The emitted X-rays are detected by an EDX fluorescence detector, while a transmission detector helps to assign the orientation of the specimen during scanning. (C) In Secondary Ion Mass Spectrometry (SIMS), the surface of the specimen is sprinkled with a focused primary ion beam and the ejected secondary ions are identified by their mass/charge ratios in a mass spectrometer. This figure part is adapted from (Fahrni, 2007).

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Q. Synchrotron X-ray Fluorescence (SXRF) Microscopy It was demonstrated that elemental mapping by SXRF microscopy is a highly sensitive method for the detection and mapping of elements such as Fe and Cu in liver sections (Fahrni, 2007; Kinoshita et al., 2010). As a powerful non-destructive analytical technique it can be used both to quantify and image the distribution of metal elements in biological samples and thus provides additional biochemical information (Fahrni, 2007; Kinoshita et al., 2010). The principle of SXRF is based on the intrinsic fluorescent properties of elements. It uses X-rays emitted from atoms excited by an external source for elemental analysis. What happens is that when an atom absorbs the source X-rays the incident radiation can cause the ejection of electrons from the inner shell of the atom resulting in electron vacancies. Thus electrons from outer shells will relax and fill the holes and emit X-ray photons. The energy of the emitted photon is dependent on the difference in energies between the shell with initial vacancy and the energy of the electron that fills the vacancy and is characteristic for each individual atom from which it originated. When measuring the intensity of the emitted energies it is possible to quantify how much from a particular element is present inside the sample (Singh et al., 2013). Multiple elements can be detected simultaneously and information regarding their spatial distribution can be obtained as long as the incident X-ray beam energy is greater than the binding energy of each element of interest. In terms of reliable quantification data the SXRF detection sensitivity can be as low as 0.1–1 mg g 1 when using standards of known concentrations (Bourassa & Miller, 2012). SXRF presents some advantages such as the sample is not damaged during the measurements, the method requires little to no chemical sample preservation, and SXRF possesses high sensitivity in the attomolar concentration range. The high flux of third generation of synchrotron sources together with the latest advances in focusing optics allow the Xray beam to be focused down to a few hundred nanometers (i.e., 0.1–1 mm) and thus provide subcellular resolution (Ralle & Lutsenko, 2009). When performing SXRF one has to use trace element-free substrates such as silicon nitride or Ultralene that represents a thin technical polymeric film (Ralle & Lutsenko, 2009) and has to keep the sample motionless during analysis so that no scattered radiation from mounting materials is produced or the beam path is obstructed (Punschon et al., 2013). A schematic view of a typical SXRF microscopy set-up is shown in Figure 4B. One important application of SXRF in biomedical research is represented by the quantitative imaging of metals in tissues. In order to detect metals in tissue it is crucial to use methods which are independent of the chemical state as well as sensitive enough to detect low concentrations of the respective metals. SXRF is able to quantitatively measure trace elements with high sensitivity and high resolution even when used on tissues. But for this application proper preparation of tissue samples is requested. Ralle and coworkers have found that the tissue morphology is properly preserved when fresh saline-perfused tissue is embedded in an optimum cutting temperature (OCT) compound and subsequently flash-frozen in dry ice-cooled isopentane. The OCT embedded block is sectioned in a cryostat and the morphology accessed from an adjacently cut, hematoxylin and eosin (H&E)-stained section (Ralle & Lutsenko, 2009). Mass Spectrometry Reviews DOI 10.1002/mas

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Another interesting application of SXRF is represented by the non-destructive visualization of metal distribution (Cu, Fe, and Zn) in brain tissue or nerve cells in the case of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis (Fahrni, 2007). In a study conducted by Miller and collaborators, the elemental mapping of b-amyloid deposits revealed the presence of hot spots of accumulated metal ions, particularly of Cu and Zn (Miller et al., 2006). Brain tissue of Alzheimer’s disease patients was analyzed by means of correlative synchrotron Fourier transform infrared micro-spectroscopy (FTIRM) and SXRF microscopy. The study demonstrated for the first time a strong spatial correlation between elevated b-sheet content in Ab plaques and accumulations of metal ions in these areas suggesting a direct association of metals and amyloid formation. The intracellular mapping of anticancer drugs can be successfully achieved by means of SXRF microscopy. This technique is uniquely suited to study the biological speciation of metal-based drugs because drug-embedded metals can be directly detected without further labeling. One seminal example is given by Hall and colleagues who studied the subcellular localization of bivalent and tetravalent platinum (Pt) complexes in a cultured ovarian cancer cell line treated with bromine containing Pt complexes (Hall et al., 2006). Cisplatin-treated sectioned cells revealed a high nuclear localization after 24 hr with a lower concentration in the cytoplasm (Hall et al., 2003). Treatment of the same cell line with three structurally related, hexa-coordinated Pt (IV) complexes yielded similar subcellular distributions compared to cisplatin, although with significant variations in the overall Pt content.

R. Secondary Ion Mass Spectrometry (SIMS) Developed in the 1960s by the French company CAMECA (www.cameca.com), the SIMS technique is used in materials and surface sciences for the analysis of solid surfaces and thin films. SIMS measures the secondary ions that are removed from the specimen after the collision with a primary ion beam. The resulted secondary ions are separated based on their mass/charge ratio within a mass spectrometer (sector field, quadrupole, or time-of-flight) in order to determine the elemental, isotopic, or molecular composition of the surface (see Fig. 4C for a schematic view of the SIMS set-up). The primary ion beam used (e.g., cesium, Cs; argon, Ar; gallium, Ga; gold, Au; bismuth, Bi; molecular oxygen, O cations) depends on the element or molecule of interest. An oxygen ion beam is usually used for the detection of most metals because it favors the formation of positive ions (Bourassa & Miller, 2012). Unlike SXRF technique SIMS is destructive to the specimen (Solon et al., 2010). SIMS is considered to be a qualitative technique, although quantitation is also possible when proper standards are being used. SIMS can operate in two different modes, static or dynamic. While static SIMS makes use of a lower intensity primary beam and stays very close to the specimen’s surface generating larger fragments such as intact molecules, the dynamic SIMS requires a higher intensity beam which goes deeper into the specimen and produces smaller fragments, making it ideal for elemental analysis (Lobinski, Moulin, & Ortega, 2006). With a detection limit of 0.01 mg g 1, and an 11

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TABLE 2. Key features of trace metal imaging techniques Imaging technique

Measurable metals

SXRF microscopy

Metals and metalloids

SIMS

Metals and metalloids

Sample (biomedical applications) Biological materials (e.g., tissues, cancer cell lines), metalbased drugs

Detection sensitivity

Spatial resolution

Quantification

100 nm

Possible