Keywords

Introduction

There are many lines of research relating to neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis (ALS), Huntington’s disease, and Creutzfeldt–Jakob disease. Frequently these are related to protein misfolding and aggregation accumulation commonly in, or near, neurons or neurologically related cells. Alzheimer’s disease relates to amyloid-β (plaque) and tau proteins (tangles). Parkinson’s disease (and Lewy body dementia, etc.) are synucleinopathies involving the abnormal accumulation of α-synuclein in the brain. Further, Huntington’s disease is related with Huntingtin protein; Creutzfeldt–Jakob disease is related to the Prion protein. Thus, unwanted protein dimers, trimers, oligomers, plaques, fibrils, and other morphologies of networks may be formed. Many researchers think these phenomena somehow are linked to the cause or result of the disease, but there are still many open questions as there is much complexity. To unearth the exact mechanism and chemical species of the disease much more basic research is needed. Other chemical aspects implicated in such disease etiology include oxidative stress, certain toxic environmental chemicals, enzyme malfunctioning, and the simple act of aging. Also, particular metals can bind into misfolded proteins preferentially (e.g., Cu2+ into histidine-rich fragments). Some researchers seek to link disease to metal ion concentration. Certain transition metals such as Cu, Zn, and Fe have normal healthful and important roles in catalytic activities or transport in the active site of the enzyme. When they are found abundantly uncomplexed and labile, toxic effects can be imparted, e.g., induction of covalent linkage between monomer proteins. Some researchers support that the abundance/activity/function of, e.g., Fe, Cu, Zn (and previously Al) are importantly related to AD and/or PD.

For metal ions in disease, there is a possibility to prepare model systems. There are of course both advantages and technical limitations in studying real living systems as well as model systems. In tissue, neuron samples, cerebrospinal fluid (CSF), e.g., some instruments can be directly analyzed. Specifically, HPLC-MS, IC, ICP-MS, MALDI-MS, ICR-MS, etc., techniques have been developed. In particular, ICP-MS is used broadly and has many advantages. Small sample volumes are allowable; short analysis times are possible (~μs for one metal in one sample); an important hyphenated utility exists with other instruments: HPLC, capillary electrophoresis, SDS–PAGE, laser ablation (LA) systems. ICP-MS has become an essential instrumental technique for determining [Mn+] in biological samples (brain, neurons, blood, CSF).

ICP-MS analysis and its practical use

Instrumental setup

Interestingly, ICP-MS is not a very new technique as it was first used commercially in 1983. A crudely estimated 8,000 or more systems are used around the world at the time of writing [1].

To break down the ICP-MS technique, five major topics need to be addressed: the sample injection portion, ICP portion, interfacing, mass separation, and detection (Scheme 1). The sample injection parts are commonly composed of a nebulizer and sample chamber. Connections can be made to many kinds of instruments, including laser ablation systems, liquid chromatography, and capillary electrophoresis. The injector system is designed to analyze liquid samples. For solids analysis, extra pure grade acid is used for dissolving the sample. Laser ablation analyzes, e.g., solid surfaces, clinical biological tissues, 1- or 2-D gel electrophoresis.

Scheme 1
scheme 1

Scheme 1

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ICP generates plasma from Ar gas and radio frequency power. Its temperature reaches 6,000–10,000 K whereupon, the sample converts into the positive ion (1+) streams (Scheme 2). Positive ion streams are directed to the interface part connecting the ICP (760 Torr) and the mass separation segments (10–6 Torr). Regulating devices of the positive ion stream include: the sampler cone, the skimmer cone, and the ion optics portion. Near vacuum is maintained (~1–2 Torr). Cooling protects the instrument from ICP. The ion stream passes through the sampler cone, skimmer cone, and ion optics. The ion optics maintains non-ionic species, such as photons and neutral species, from reaching the detector, which would otherwise cause artifacts [1].

Scheme 2
scheme 2

Scheme 2

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Positive ion streams contain interfering molecules produced by Ar+ or O. So, the dynamic reaction cell (DRC) or collision cell separates the analytes from the interferents [2]. Compared with the ICP-OES method, eliminating interference molecules is one of the most important aspects in ICP-MS analysis [3]. There are different hyphenated instruments for mass separation, time of flight (TOF), double focusing sector field (SF), quadrupole, etc. The detector quantifies and amplifies the signal obtained from various concentrations: very low (~ppq) to relatively high (~ppm). Commercially used apparati include the channel electron multiplier, Faraday cup, and discrete dynode electron multiplier [1].

Elements according to their limits of detection and normative values

ICP-MS is engineered to accommodate the analysis of more than 70 elements. Commonly, almost all are positively charged elements; some are commonly negatively charged elements (e.g., halogens, chalcogenides), etc. [1]. Experimentally, elemental detection is limited; elements are grouped according to the common range of different detection limits, provided below (Table 1) [1]. Most elements (65) can be detected below 1 ppt. For Be, B, Hg, this detection limit is 1 ~ 100 ppt. Si, P, and S have detection limits of 0.1 ~ 1 ppb; Br and I have limits of 1 ~ 50 ppb.

Table 1 Groups of elements (given as their symbols) according to their limits of detection for the ICP-MS technique
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Ions implicated in molecular neurodegeneration such as Cun+, Zn2+, Fen+ have been analyzed by ICP-MS; these ions are usefully found in the body above the limits of detection. A range of normative values of metal ions are provided below in Table 2. Levels of these elements in, e.g., CSF for healthy human subjects are usually in the range of 20–30 μg L−1 [4], a range appropriate for ICP-MS analysis.

Table 2 Trace element concentrations of Cu, Fe, and Zn found for normal human subjects
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Ions are separated by their mass-to-charge ratio, thus, there is a difference between detecting an ion or a neutral atom. Various mass separators can be used including the TOF, sector field double focusing, quadrupole, etc. To detect neutral atoms, a different strategy is required involving spectroscopic methods, e.g., optical emission spectroscopy (OES), energy dispersive spectroscopy (EDS), nuclear magnetic resonance (NMR) spectroscopy, etc. The elemental precision for neutral atom detection is lower than ion detection [1].

Comparison with other forms of inorganic instrumental analysis

There are several atomic spectroscopic techniques, such as flame atomic absorption, atomic emission spectroscopy (Fig. 1). Flame atomic absorption is a single-element technique. The flame is generated from a graphite furnace (2,500–4,000 K). When the sample enters the flame via the instrumental nebulizer, the ground state atom absorbs at an element-specific wavelength (λ abs) measured and detected using a monochromator, photomultiplier, and a solid-state detector in order to calculate the concentration of element. This technique while being fast, is also relatively inexpensive, and easy to handle without excessive technical training. Flame AAS can be hyphenated with HPLC. However, the sensitivity here is somewhat low. Detection limits at the ppm level are common, meaning that ICP is superior in sensitivity [1, 5–7].

Fig. 1
figure 1

Schematic illustration of the instrumental setup for atomic absorption and emission, and atomic mass spectrometry. Adapted from Ref. [1]

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Atomic emission or OES usually involves the use of an inductively coupled plasma source, 6,000–7,000 K. ICP-OES is multi-elemental. In plasma, electrons in sample atoms are excited to higher energy. The amount of light (wavelength signal λ em) emitted when the excited electrons return to the ground state is then detected. The intensity and integration of the spectral signal gives the concentration of the aforementioned elements. Radial and axial types are used: in the axial type, the plasma is horizontally positioned, so more photons can be procured than for the radial type (improvement by 2–10 times in detection limit) [1]. Because a cone is not used, the tolerance to matrixes is good, and it can more easily be “hyphenated,” e.g., with HPLC. Further, it is possible to detect sulfur or phosphorus (see Table 1), because interfering molecules do not need to be removed, when compared to ICP-MS [6].

Sample preparation and analysis

Since ICP-MS can help analyze various sample types such as fluid, tissue, and tissue for bioimaging, care in sample preparation is very important. To protect the sample from metal or element contamination is vital; thus exquisite cleaning of instrumental components such as tubing is very important. For this procedure, high- or extra-pure grade acids (nitric acid) are used and a clean room is required [8]. Plasticware is often used for sampling, transferring, and storage [8]. Glass is generally not used so to prevent leeching of metal ions out of the glass matrix [9]. Usually apparati are made of quartz, Teflon, polyethylene (PE), polypropylene (PP), polysulfide [10], and polytetrafluoroethylene (PTFE) [1].

Fluid analysis

Analyte samples are stored at −20 °C [4]. In some cases, digestion by microwave irradiation is needed. Stainless steel needles are used to sample biological fluids, which is transferred into precleaned polymer tubing (vide supra). Samples in Pyrex tubes are placed into steel bombs via Pyrex tubes and sealed with a Teflon lid [11]. In this way, some protein in the buffer system can be directly analyzed [12].

Tissue analysis

Sample containers are cleaned with aqueous HNO3 for several hours prior to use. Tissues are cut in thin slices on an acid-washed polyethylene surface by an acid-washed quartz knife. Samples can then be homogenized by motor-driven Teflon pestles or dried via a vacuum drier or freeze-drier. In some cases, the samples may be digested in a sealed Teflon “bomb” in a microwave oven in acidic solution. Dilution is made with ultra pure water [13, 14].

Bioimaging analysis

Usually, biological sectioning is performed with a ceramic or stainless steel knife. To obtain samples of micrometer thickness, special instrumental dissectors, microtomes, or cryo-cutting tools are used. Sectioned tissues are mounted and dried on sodium-free glass slides [15–17].

Neurodegenerative diseases and metal ion analysis with ICP-MS

Neurodegenerative diseases

Alzheimer’s disease

AD is progressive and irreversible and is a type of dementia that involves eventually grave injury to the brain characterized by extensive neuronal death. Symptoms include memory loss and personality changes. “Mild cognitive impairment” (MCI) is the name given to the earliest detectable stage of AD. Major physiological/pathological characteristics include extracellular senile plaques (amyloid) and intra-neuronal neurofibrillary tangles (tau) in the brain. Senile plaques arise from Aβ deposition from excessive scission of the amyloid precursor protein (APP), and neurofibrillary tangles from “hyperphosphorylated” tau.

Metal ions are implicated in disease aetiology (vide supra). Metal-catalyzed oxidations relating to transition metal ions of Cu, Fe, and Zn may lead to mitochondrial dysfunction and molecular processes of aging. Copper can enter the cell via ways including the copper transporter 1 (Ctr1), specific for Cu (I), in the intestinal absorptive cell. Inside the cell, copper ions are transferred to certain cellular organelles or molecules including the Golgi complex, superoxide dismutase (SOD), and the mitochondria. Processes are mediated by metallochaperones. These are specialized proteins that can protect metals from scavengers: the trans-Golgi network (Atx1), copper chaperones for SOD (CCS), and cytochrome c oxidase (Cox17) in the mitochondria [18]. Genetically, MURR1 mouse (U2af1-rs1), COMMD1 (copper metabolism gen MURR1) were studied; these species were discovered to be important for regulation of copper concentration. COMMD1 can bind to copper ions to induce conformational changes; this relates to the interaction with other regulatory proteins. Metallothioneins (MTs) also participate in copper regulation. Four distinct MTs are known. MT1–MT3 are found primarily in the brain; MT4 exists outside the brain. These molecules are cysteine (cys)-rich and can bind a range of ions (e.g., Cu, Zn). If copper regulation is not efficient, these ions impart a cytotoxic effect. Copper incorporation into the cell sometimes fails in such states as Menkes disease (copper deficiency). In the case of the failure of copper to become excreted from the cell, this may signal Wilson’s disease (copper accumulation). When Cu(II) levels are not controlled in the cell, Cu(II) can bind to the Aβ protein, which may turn on production of hydrogen peroxide. Cu(II) reduction to Cu(I) occurs through the use of biological reducing agents such as NADH, NADPH, ascorbate, catecholamines, and cholesterol. The Aβ–Cu(I) metal complex reacts with H2O2 to give hydroxyl radical and reactive oxygen species [18]. These reactions are known as the Haber–Weiss or Fenton reactions [18–20]. Hydrogen peroxide can be the source of various reactive oxygen species [21] that can induce covalent links between proteins (protein–protein cross-links) and oxidation at sites in the protein backbone [20] (Fig. 2).

Fig. 2
figure 2

Formation of cross-linked Aβ using copper. Adapted from Ref. [20]

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Zn2+ homeostasis is understood to be highly regulated; but an understanding of many aspects of this regulation are still sought. Various specialized biomolecules such as members of the Zn transporter (ZnT) (more than 10), ZIP (Zn(II)-regulated metal transporters, iron-regulated metal transporter-like proteins) (15 members), and metallothionein (3 isoforms) [18]. Usually Zn2+ efflux is related with the action of zinc transporter, Na+/Zn2+ exchanger, and ZIP [22]. Free Zn2+ can induce toxic effects. When Zn2+ enters the postsynaptic neuronal region through Ca2+–A/K channels, it is observed to generate oxygen species rapidly [23]. These ROS can induce mitochondrial dysfunction and oxidative stress to increase the activity of neuronal nitric oxide synthase and NADPH oxidase, which in turn generates peroxynitrite (O=NOO−). This allows mitochondria to decrease the ability of Zn2+ sequestration in the cytosol. This “positive feedback” is thought to induce cellular apoptosis or necrosis; eventually neuronal cell death ensues [18]. Zn2+ buffering in the cell is regulated by metallothioneins. MTs are composed of 61–68 amino acids, which have a sequence of 20 cysteine (cys) residues that is highly conserved. Such Zn2+ sequestration from the cytosole is controlled by mitochondria [24].

The physiological role of iron in the brain is often considered with such topics as (1) embryonic neuronal development, (2) myelin formation, (3) production of neurotransmitters, (4) DNA synthesis, (5) phosphorylation and ATP synthesis [18, 25, 26]. Iron homeostasis is regulated via Transferrin, Transferrin receptors, divalent metal transporters, the ferritin protein, ferroportins, mitochondria, endosomes, etc. Iron trafficking is mediated by Transferrin, Transferrin receptors, cubilin, divalent metal transporter, and the hemoglobin scavenger receptor (CD163). Inside the cell, iron levels are regulated by endosomes and mitochondria. Iron is usually stored in ferritin which can store up to ~4,500 iron ions. This protein is also a highly symmetrically designed container entity of 24 light, and heavy subunits. Iron release is mediated by ferroportin [27]. Iron toxicity largely occurs by oxidative stress based on the Fenton reaction. ROS react with membranes, proteins, and DNA strands alike; the expression of p53 and caspase 3 are stimulated. Cytochrome c is released from mitochondria; eventually, cell apoptosis or necrosis ensues [28].

Parkinson’s disease

Parkinson’s disease is the most common neurodegenerative disorder affecting motor skills: a sufferer may experience muscle rigidity, bradykinesia (Greek etymology: bradys = slow, kinesis = movement), resting tremor, as well as cognitive impairment. Progressive dopaminergic neuronal cell death occurs in the substantia nigra (SN) region in the brain [29]; SN means black substance. Pathological hallmarks include Lewy bodies and Lewy neuritis, which are eosinophilic and round cytoplasmic inclusion bodies in which the main component protein is α-synuclein filaments. α-Synuclein (AS) is strongly, but not exclusively, linked to Parkinson’s disease. Simplistically, AS is to PD as Aβ is to AD. There is also LBD or lewy body dementia. The oligomer form of α-synuclein is known to be more toxic to the neuronal cell than ones in fibrillar form [30]. α-Synuclein is one of the most abundant proteins in the postsynaptic protein. It is composed of ~140 amino acids and usually found in its unfolded form (in the cytosol) [31]. There are many areas of research regarding aggregation of α-synuclein, metal-catalyzed oxidation [32]; certain toxic aldehydes have also been considered as being linked to disease as well (4-hydroxynonenal [33], oxidized cholesterol [34], 3,4-dihydroxyphenylacetaldehyde (DOPAL)) [35]. Long-term exposure to transition metals, or aging, stress can also influence the homeostasis of metals. Uncontrolled iron and copper concentration can generate reactive oxygen species that can allow α-synuclein to cross-link; this may produce aggregates of oligomers and fibrils. Dysregulated free metal ions in neuronal cells can influence mitochondrial dysfunction, effect changes in antioxidant systems of monoamine oxidase type-B (MAO-B), SOD activity, and allow for abnormal interactions with neuromelanin with iron. Some key reactions in mitochondrial respiration involve Fenton chemistry and dopamine oxidation (Scheme 3) [36].

Scheme 3
scheme 3

Scheme 3

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Menkes and Wilson's Diseases

Menkes disease and Wilson’s disease are disorders involving atypical copper transport and homeostasis, are genetic. They exhibit marked but different neurological manifestations. Mutations in the P-type copper transporting ATPases, ATP7a (MNKP, Menkes protein) exist in the case of Menkes disease; Mutations in ATP7b (WNDP, Wilson protein) are found in the case of Wilson’s disease. Significant disruption in copper trafficking results [19, 37]. In Menkes disease, copper blood–brain barrier (BBB) penetration is inhibited leading to copper deficiency in the body. Wilson’s disease is caused by copper overload: whole-body copper accumulation leads to brain/liver copper deficiency. Further, there is holoceruloplasmin biosynthesis and a marked impairment in biliary copper excretion [18, 26].

Amyotrophic Lateral Sclerosis

ALS (sometimes referred to as Lou Gherig’s disease) is a progressive and fatal neurodegenerative disease caused by the degeneration of motor neurons. Certain prominent cases include that of Scientist Stephen Hawking. Approximately 10 % of cases are linked by genetic factors; these incidences are termed as cases of familial ALS (fALS). Other cases (the majority) are termed “sporadic” (sALS) [19]. Approximately 20 % of fALS cases are caused by inherited mutations in the gene encoding Superoxide Dismutase 1 (SOD1). SOD1 is homodimeric (2 × 153 AA) and normally functions as an antioxidant by detoxifying the potentially damaging superoxide radical anion. Its important function relates to its structure: each subunit is composed of an eight-stranded “Greek key” β-barrel fold; each one binds one zinc and one copper ion; there is an intrasubunit disulfide bond as well. Disproportionation of the superoxide anion (O ċ−2 ) is effected catalytically at the copper site; here, alternating cycles of reduction and oxidation exist. Copper and Zinc in the enzyme play, not only catalytic roles, but are also involved in preserving correct protein folding and in retaining “negative design.” Loss of metal ion (demetallation) may induce protein misfolding and aggregation [19, 38–40].

Prion diseases

Prion diseases, also known as transmissible spongiform encephalopathies, are degenerative neurological diseases that relate to a “contagious” protein and its misfolding. These disorders include the classic Creutzfeldt–Jakob disease, Gerstmann–Sträussler–Scheinker syndrome, fatal familial insomnia as well as Kuru (New Gunea) in humans. Prion diseases are associated with the accumulation of misfolded cellular prion protein (PrPC); PrP readily binds copper ion. While the exact role of PrP is still unknown, two main biological functions of the prion protein appear to be copper transport and antioxidative activity. Besides copper, an imbalance of other metals (ions) such as iron, manganese, and zinc is also associated with prion diseases [19]. Many investigations of the potential pathogenic role of manganese, including conformational changes of protein and non-homeostatic redox activity, have been made [41, 42].

Huntington’s disease

Huntington’s disease is a genetic disorder originating from the mutation in gene coding for Huntingtin (Htt) [43]. However, the exact function of Htt, as with many other proteins mentioned herein, is still unclear, the protein is thought to impact myelin integrity and plays a role, albeit indirectly, in the striatal iron accumulation observed in HD. Proposed mechanisms where iron homeostasis might lead to HD have been previously reviewed [44]. Copper imbalance is also observed in HD patients. A contribution of copper–protein interaction to HD pathogenesis has also been studied [45].

Tissue analysis

In many tissue portions, there is much research and analysis about the concentration of metals in these tissues—the metal concentration can be telling about pathology. There are carefully classified regions of the human brain (Fig. 3) below the broad regions of the cortex and medulla (Fig. 3). Subdivisions of the brain include the hindbrain (Myelencephalon, Metencephalon), Midbrain, Forebrain (Diencephalon, Epithalamus, Third ventricle, Thalamus, Hypothalamus, Subthalamus, Pituitary gland, Telencephalon, White matter, Rhinencephalon, Lateral ventricles, and Cerebral cortex).

Fig. 3
figure 3

Some regions of the human brain

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Inductively coupled plasma mass spectrometry (ICP-MS) can be used in the assessment of trace element brain tissue to assess regional concentrations. These multi-element analyses help quantity of which metal ions and can be correlated to neurodegenerative disorders. Concentrations of 16 elements (Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Hg, Pb) were measured in three regions of the human brain (frontal cortex, frontal white matter, and cerebellum) using ICP-MS by Ejima et al. [46]. These sample analyses from subjects who had died and did not possess incidence of neurological disease were compared with those of the tissues of the patient with FALS. These data and those obtained by other techniques such as neutron activation analysis (NAA) and ICP-AES were in good agreement. Also, the higher concentration of Mn in the samples of the ALS patient than in the controls was observed.

The ICP-MS technique has also been widely used in the determination of the concentration of the certain metals in studies relating to AD. The concentration of Zn and Cd have been estimated through analysis of human brain tissue samples from Alzheimer’s disease (AD) subjects (brain areas: superior frontal gyrus, superior parietal gyrus, medial temporal gyrus, hippocampus, and thalamus (both hemispheres)) and compared with those for control subjects [47]. Values here are given for all regions of the brain, including both hemispheres. The value ranges of the arithmetic means with standard deviations were reported in ng g−1 for Cd and mg g−1 for Zn. Concentrations of Cd and Zn in “normal” samples are obtained in the 5–145 ng g−1 and 6.4–29.3 mg g−1 range with arithmetic means of 26.7 ± 22.2 and 13.8 ± 4.0, respectively. The values for Alzheimer’s disease samples were in the 4–104 ng g−1 range and 32.1 ± 19.0 ng g−1 for Cd and Zn, respectively, the arithmetic mean for Cd and Zn were 7.1–19.4 mg g−1 and 13.8 ± 2.7, respectively. Importantly, differences in element concentration are expected when considering brain regions, disease stage, age, and gender.

In a paper by Corrigan et al., 38 different elements were analyzed by NAA and ICP-MS [48, 49]. Brain tissue acquired post-mortem from 11 human subjects previously diagnosed with Alzheimer’s disease, and from six controls were used (brain region: temporal lobe and frontal cortex lobe, caudate nucleus and putamen). Elevated tissue concentrations of aluminum, silicon, and calcium were reported (area: cortical areas, basal ganglia) while zinc, selenium, cesium, and cerium were lower. Plaque cores themselves can be successfully analyzed by ICP-MS. Here, it was combined with flow injection (FI) for selective extraction. Concentrations of several plaque metal samples were measured, including Cr, Mn, Ni, Cu, and Pb (0.2–0.8 mg L−1) and Al, Fe, and Zn (2–20 mg L−1) [14].

Furthermore, metal ions of Mo and especially Mn, have attracted much recent attention in the AD [50, 51], Parkinson’s disease [43, 52, 53], ALS [13], and prion disease research fields [41, 42]. In one study by Csaszma et al. where a total of 5 brain regions were studied, the mean concentration of Mn was found to be in the range of 1.1–1.3 ppm, whereas that for Mo was between 90 and 150 ppb for dry weight tissue (both control and AD subject). The high concentrations of Mo and Mn 330 ± 42 (ppb) and 2.90 ± 0.07 (ppm), respectively, were found for AD patients in the putamen brain region (dry weight) [50]. Higher concentrations of manganese, as well as Si, Sn, and Al, in the parietal cortex of the AD brain compared to control values were measured by the research group of Srivastava et al. [51]. Concentrations in the non-metal B and other ions of metals such as Na, Te, Cr, Fe showed moderate increases in the same brain region. Changes in Mn, Fe, Cu, and Zn concentration in rat brain where Parkinson's disease was induced by 6-hydroxydopamine (6-OHDA)-induced parkinsonian symptoms were observed in all regions that lay along the dopaminergic pathway [43]. Measurements were performed via ICP-MS at 1, 3, 7, 10, 14, and 21 days after treatment with 6-OHDA. Stable 55Mn, 56Fe, 63Cu, and 64Zn isotopes were measured simultaneously with the procedural detection limits of 8, 70, 27, and 30 pg g−1, respectively. In studies relating to the effects of Mn exposure on neurotoxicity, and the subsequent exacerbation of parkinsonism, concentrations of manganese in the whole brain were measured with an accuracy of an average 94 %; here the analytical detection limit is 0.16 ppb [53]. A study of concentration profiles of Fe and Cu upon Mn exposure in brain tissue for juvenile and adult mice was made [52]. The influence of manganese uptake on the pathogenesis of prion disease was also investigated [42]. Data obtained by ICP-MS indicate the increase in intracellular Mn levels for the mouse brain slice cultures upon chronic Mn exposure, which did not affect levels of Cu, Fe, Zn significantly altered by the aforesaid manganese treatment. In an in vivo chelation therapy study, decreases of Mn levels by up to 50 % in treated mice compared with untreated controls was determined by ICP-MS [41]. Interestingly, copper, iron, zinc, and cobalt levels remained unchanged.

Concentrations of several metals Rb, V, Mn, Fe, Co, Cu, Zn, and Cd were estimated in formalin-fixed brain tissue from subjects (of Guam) who suffered from ALS; four subjects were confirmed to have Parkinsonism–Dementia Complex (PDC); five served as control subjects [13]. Two metals (Zn and Cd) were reported at different values; for other metals, no significant differences were observed. An increase in the concentration of Zn for both subject groups was observed (gray and white matter). Cadmium gives higher values in ALS, but not in PDC subjects. Alkali metal ions (Li+, Na+, K+, Rb+, and Cs+) also can be detected in several AD brain parts [54]. Mg content in AD brain was studied by Andrasi et al. [55]. A decrease in Mn concentration was detected by several instrumental methods including ICP-MS, ICP-AES, and NAA.

Environmental pollutants including heavy metals can enter the food chain and eventually contaminate the human central nervous system (CNS) [56, 57]. Ni and V exposure on nasal and brain areas in dogs was also studied and reported [57]. ICP-MS data showed higher [V] in exposed animals over those in control animals (0.36 ± 0.3 μg g−1 vs. 0.19 ± 0.17 μg g−1, respectively).

Fluid analysis

Fluid analysis is very straightforward in ICP-MS. Usually it is possible to analyze the liquid sample directly, e.g., in its neat liquor form. In several studies, the involvement of metal ions in pathogenesis of neurodegenerative diseases have been supported by analysis of body fluids. Certain important fluids (CSF, blood, etc.) relate either directly or indirectly to CNS diseases. ICP-MS widely used for quantification of several metal ions in CSF, urine, serum, and whole blood. In most cases, Sector Field Inductively Coupled Plasma Mass Spectrometry (SF-ICP-MS) was used to avoid several disadvantages of the conventional quadrupole mass filter (Q-ICP-MS): these include signal interference during the experiment originating from plasma, water, reagents, and matrix of the biological samples used [58]. Samples of CSF from subjects of a large group of PD patients (n = 42) was analyzed for metal content by the research group of Alimonti in 2007 [8]. A total of 20 metallic elements (Li, Be, Sr, Ba, Zr, V, Cr, Mn, Co, Ni, Mo, W, Cd, Hg, Al, Sn, Sb, Bi, Pb, and Tl) were detected by SF-ICP-MS. Additionally, 6 elements (Si, Mg, Ca, Fe, Cu, and Zn) were quantified by ICP-AES. Researchers represented values from a mixture of transition metals and others statistically for PD subjects and controls and the plot of element discrimination between control and patient: data indicated abnormally low levels of Si, Cr, Fe, Co, Sn, and Pb in the CSF of PD patients compared to values from control samples. Previously, the same research group detected significantly increased concentrations of Pb and V in blood and urine (p ≤ 0.03, for both metals) and reduced levels of Al, Cd, Hg, and Pb in serum and Cr, Co, Cd, Hg, Pb in CSF (p ≤ 0.003) [59]. The detection limits for most of the elements range from 0.001 ng ml−1 for Cd to 0.05 ng ml−1 for Al, and were satisfactory and suitable for the intended purposes. Here, the applied ICP-MS method provided good accuracy; for example, in urine this value ranged from 92.4 % for Ni to 105 % for Hg; in the case of serum from 97.8 % for Mn to 104 % for Al; and in blood it varied between 97.0 % for Cr and 103 % for Pb.

Forte et al. determined concentrations of [Al] and [Mn] in urine, blood, and CSF for 26 PD human cases [60]. Both metals gave no statistical difference in concentration levels for patients and controls in any of the analyzed fluids. One exception was the [Al] in blood serum; this was lower in PD patients and a slight decrease, albeit not statistically relevant, in manganese concentration for whole blood and CSF was also found for PD patients. A similar trend for Al was reported by Bocca et al. in 2006 [61]. Here, concentration values of 3.0 ± 1.8 in PD patients vs. 5.3 ± 3.4ng ml−1 for the control group in serum and 11.7 ± 7.05 vs. 20.7 ± 12.6 ng ml−1 in urine, respectively, were detected. In a separate study, the analysis of 31 elements in samples of serum for 45 PD patients and 42 controls showed that [Cu], [Al], [Mn], and [Zn] are central ions in separating PD cases from control cases [62]. Concentrations of 31 elements in CSF, blood plasma, and whole blood were determined in living human subjects suffering from (1) multiple sclerosis [63], (2) so-called “Skogholt’s” disease (a demyelinating disorder of both the central and the peripheral nervous system involving adult onset) [64], and (3) those from a control group [4]. Through analysis with SF-ICP-MS, a threefold increase of [Cu] and [Fe], and a twofold increase in [Zn] were detected in the CSF of “Skogholt” disease patients compared to those of controls. Other elements such as Rb, Co, P, S, Th, and Se were also found at higher concentration levels in “Skogholt” subjects. Interestingly, the decrease of Mo, Cd, Hg, and Tl levels in “Skogholt” and multiple sclerosis (MS) samples compared to the control group values.

Recently, Isao Hozumi's research group studied biological metal ions, Cu, Fe, Zn, Mg, Mn, in the CSF of patients with ALS (52 people), AD (21 people), and PD (20 people) subjects through the use of ICP-MS [65]. Cu and Zn in AD, Cu, Zn, and Mn in PD, Cu, Zn, Mg, and Fe in ALS were elevated compared to normative values. In particular, Cu ions (18.8 ng/ml) of Parkinson’s disease patients showed increased levels over control values (10.2 ng/ml).

Additionally, Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr, Mo, Cd, Sn, Sb, Cs, Hg, and Pb were measured from the CSF and plasma of AD patients by the group of Gerhardsson. Quotients of metal contents were assessed between CSF and plasma. The quotients of Mn, Rb, Sb, Pb, and Hg were low, whereas that for Co (Q Co = 0.09) was high in AD subjects compared to those of healthy controls (Q Co = 0.06) [66].

Bioimaging of metal ions in brain samples and the advent of LA-ICP-MS

In recent years, bioimaging and metal distribution of the brain are researched by way of laser ablation ICP-MS (LA-ICP-MS). LA-ICP-MS is a widely used analytical spectrometric imaging technique; trace metal ion concentration/distribution determinations in brain tissues are made via imaging. Examples of practical applications of LA-ICP-MS in neurodegenerative disease studies have been previously published [67, 68]. In comparison to the tissue or fluid case, it is possible to determine the microstructure of the sample, as well as the metal distribution. Zoriy et al. analyzed human brain tissue for 13 C+, Cu, and Zn distributions; five adjacent sections were analyzed. The reproducibility was 5.4–6.5 %, 5.8–8.2 %, and 5.1–6.7 %, respectively, for aforementioned elements [15]. Becker et al. investigated the insular region and tissue from brain tumor to detect distribution of Cu, Zn, and certain other elements. The detection limits were 340 ng g−1 and 140 ng g−1, respectively [69].

In a Parkinson’s disease study of the distribution of several metal ions (iron, copper, and zinc) in a (Parkinson) mouse model brain was compared with control values [67]. The images obtained by LA-ICP-MS clearly show significantly higher iron content in the substantia nigra (SN) brain region in the Parkinson brain in control samples. Through similar research conducted by Matusch et al. [16], quantitative images of Mn, Fe, Cu, and Zn were generated in the Parkinson brain. This “time-course” series of images involved tissue exposure with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The most pronounced changes were observed for Cu and Fe (Fig. 4). There was an increase of Cu (38 %, versus control) in the periventricular zone at 28 days, and of Fe (38 % and 39 %) in the interpeduncular nucleus at a time of 7 and 28 days, respectively.

Fig. 4
figure 4

Images of brain cross-sections and analysis of Cu and Fe content for the control (top row), 2 h (second row), 7 days (third row), and 28 days (bottom row) after the last of five daily MPTP injections. Reprinted with permission from Ref. [16] (License from Rightslink/Copyright Clearance Center. Licensee: David G. Churchill, License Date: Nov 18, 2010, License Number: 2551800410899, Type Of Use: reuse in a journal/magazine)

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A report by Hare et al. [70] discussed an observed increase in the concentration of 56Fe in a 6-hydroxydopamine (6-OHDA) lesion mouse brain model within the substantia nigra (SN) compared with control animal subjects. The quantitative Cu distribution was analyzed for 2- and 14-month-old mice; this was done to probe the notions that (1) neurodegenerative diseases are age-related diseases and that (2) copper may play an important role in the brain in aging [71]. While total Cu content in brain after intravenous (i.v.) administration of 67Cu increased in both cases; however, in the striatum and ventral cortex, Cu showed an approximately 50 % reduction in 14-month-old mice compared to those who were 2 months old (Fig. 5).

Fig. 5
figure 5

Images demonstrating distributions of Cu in cross-sections of brains of 2- and 14-month-old mice. Reprinted with permission from Ref. [71]

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Iron and zinc distributions also were investigated [72, 73]. Zinc does not give any changes, whereas Fe gives increased values in the Substantia Nigra, the thalamus, and the CA1 region of the hippocampus. In an Alzheimer’s disease study, LA-ICP-MS was also used for metal distribution and also in imaging the localization of Aβ peptide in brain tissue. For the imaging of amyloid plaques, 60Ni and 153Eu radioisotopes were used [17]. Concentrations of metal ions were different in plaque than in non-plaque tissue samples; important plaque: non-plaque ratios were calculated, and some are provided as follows: 3.5:1 for Mg, ~3:1 for Ca, “2:1 for Fe, 3.5:1 for Cu, and 2:1 for Zn.”

Additionally, tables are provided regarding the contents of Cu, Zn, and Fe in brain tissues from subjects who were normal or suffered from neurodegenerative diseases (Tables 3, 4, 5, 6, and 7).

Table 3 The contents of Cu, Zn, and Fe in normal people with ICP-MS or related instruments
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Table 4 The contents of Cu, Zn, and Fe in AD patients with ICP-MS or related instruments
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Table 5 The contents of Cu, Zn, and Fe in PD patients with ICP-MS or related instruments
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Table 6 The contents of Cu, Zn, and Fe in ALS patients with ICP-MS or related instruments
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Table 7 The contents of Cu, Zn, and Fe in other neurodenerative patients with ICP-MS or related instruments
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Protein analysis applications with metals and ICP-MS

Isotope Dilution Analysis

Isotope Dilution Analysis (IDA) is an important technique that allows the exact concentration of a sample to be obtained by adding an isotopic sample of exact isotope ratio or concentration to an unknown sample (spiking). The experimental isotope ratio of the mixture is then obtained. Advantages here include the fact that results are not influenced by the (1) mode of sample preparation, (2) various instrumental instabilities, (3) signal drift, (4) matrix effects, and (5) sample loss upon equilibrium mixing between the sample and the spike [77]. The two modes for this method are “species specific” and “species unspecific.” In the specific case, the sample species is the same as the isotope chemical compound. It is thus necessary to know the exact composition and structure of the sample. The isotope sample can be added at the beginning point of the analytical procedure. The “species unspecific” case allows the sample species to be different from the isotope “spike.” This method can be used when the structure and composition of the sample species are not accurately known. Often it is expensive to purchase certain commercially available reagents or even synthesize them. In this case, the isotope “spike” could be added to the sample after the sample preparation and treatment are effected, e.g., performing chromatography immediately before the ionization process [77]. The IDA method does have some challenges related with (1) instruments, (2) spectral interference, (3) detection dead time, (4) mass discrimination, etc. Spectral interference occurs from the same atomic mass molecules by H, O, Ar, and so on. A double-focusing spectrometer operating at high resolution helps overcome this problem. For the quadrupole instrument, there is a removable apparatus: a DRC or collision reaction cell (CRC). For DRC’s reactive gases such as O2, CO, NH3, and CH4 are used among others; the CRC uses colliding gases, such as H2 and He [77]. At high counting rates (106 + cps) an electron multiplier-type detector has “dead time:” the signal cannot be processed completely. In practice, a high abundance state of isotope can be influenced by “dead time” [77]. Heavier elements may be detected more precisely than lighter elements because of the space-charge effect; heavy isotopes show a higher intensity than light isotopes. This can be corrected through known methodolgy with certified isotopic composition materials [77]. In a report by Jakubowski et al. the investigators tried, via iodine labeling, to quantify bovine serum albumin (BSA), chicken egg white lysozyme and porcine gastric mucosa protein where an iodo-histidine or -tyrosine residue was incorporated into the protein. The stable isotope 127I was detected via ICP-MS. The investigation range was from 0.015 (BSA) to 105 pmol (lysozyme) [78]. So the ICP-MS is essential for IDA. This method can decrease the discrepancy of the matrix effect or sample preparation [77, 79]. Becker et al. performed isotope dilution of 63Cu+, 65Cu+ in analyzing human brain fluids [80], and studied 65Cu, 67Zn, and 54Fe for tissue of Alzheimer’s disease brain [81]. Heumann et al. discussed the possibility of using isotope dilution method with ICP-MS as a routinely for trace elements. Hyphenating, e.g., with liquid chromatography, and coupling of gases can help in the difficulty of stemming from the lack of commercially available isotope-labeled “spike.” If the isotope can be generated in a closed system the “spike” will not be lost. Isotope dilution techniques are useful for the accuracy of detecting MeHg+ and Cr6+ [82].

Immunoassay techniques

We will now describe how protein quantification can be more thoroughly exploited with ICP-MS. There are techniques for protein quantification via analyzing metal ions tagged with antibodies (Scheme 4); here, the metal is attached covalently to an antibody. Lou et al. reported the use of certain kinds of polymers bearing metal-chelating functional groups; the S–S cross-linked bond in the protein in question allowed for the reaction to polymers. Upon conjugation with the metal complex, the purified metal ion attached antibodies were analyzed [83] (see Scheme 5).

Scheme 4
scheme 4

Scheme 4

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Scheme 5
scheme 5

Scheme 5

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In 2008, Razumienko et al. compared ICP-MS-based immuno-protein quantification with conventional immunoassays (colorimetric and indirect ELISA). Samples included protein (hPDGF–AA), p53 in cell lines (A431, 293T, KG1a, K562). Whether Tb- or Tm-attached antibodies were used, results of analysis were similar with conventional colorimetric ELISA or the indirect ELISA [84]. Next, specific element-tagged antibody methods were reported by Banarov et al. in 2002. Methods of centrifugal filtration, Protein A affinity, size-exclusion gel filtration, and ELISA with ICP-MS were connected through the use of specific element tagged antibodies. The investigators reported that levels of target protein (0.1–0.5 ng ml−1) can be measured, involving a linear response to over three orders of magnitude. The ICP-MS hyphenated method revealed more precise results obtained more conveniently [85].

Hyphenated instrumental applications

There are related hyphenated instruments whose designs are often considered in the realm of engineering but can also be briefly mentioned here. Another mass separator, the sector-field analyzer can be used and shown to give higher accuracy. But in this case, a prospective high instrument cost, as well as significant maintenance costs are expected. When using instruments of the hyphenated design, it is required to optimize the replicate time, “dwell time,” number of sweeps per readings, and other parameters between instruments and the ICP-MS technique [6]. Helfrich et al. succeeded in coupling the gel electrophoresis technique to that of the ICP-SFMS. They attempted to detect the 32S+ and 31P+ of α- and β-caseins so to better understand phosphorylation levels. Results obtained matched well with anticipated values [86].

Conclusion

In this chapter, we summarized the utility of ICP-MS and related techniques for determining trace element concentrations in tissues related to neurodegenerative disease studies. The practical use of ICP-MS was also presented. As discussed, the accurate determination of trace elemental distributions in cases where AD or PD were diagnosed, allow for a better understanding of such diseases and relates to the sensitivity and scope of the technique. Analytes detected involved tissues and fluids. This chapter reviewed the instrumental setup, elements and their detection limits, a brief comparison of ICP-MS with other inorganic analysis instruments, and sample preparation and analysis methods. Also, material about neurodegenerative diseases and metal ion analysis were presented in the context of the ICP-MS technique. The reader should find this chapter has included certain important biophysical, analytical, and clinical studies that underscore the practical use and development of ICP. This chapter while current through the summer of 2011, is not meant to be exhaustive but was meant to be user-friendly. Along with the inclusion of various important studies, it also mentions related techniques, recent improvements, and limitations of current techniques.