The importance of speciation analysis in neurodegeneration research

Highlights

• The review shows a new trend of combining element speciation with neurology.

• The findings for cerebrospinal fluid and brain (beyond neural barrier) are discussed.

• The role of trace elements/-species in neurodegeneration is summarized in brief.

• The focus is on Al, As, Cu, Fe, Hg, Mn, and Se.

• Quality control and species stability are stressed.

Abstract

Element speciation offers deeper insight into the molecular mechanisms of disease by determining element species pattern. Thus, having great potential for investigating neurodegeneration in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and mild cognitive impairment, speciation is increasingly considered in epidemiological or clinical neurological studies. This review analyses recent speciation findings in neurodegeneration research, concentrating on measurements in cerebrospinal fluid and brain. Elements considered are aluminum, arsenic, copper, iron, mercury, manganese, selenium and zinc. Also interactions of trace element species are discussed briefly. Typically, hyphenated techniques are used in neurodegeneration speciation studies. The results allow sorting-out less important species from compounds significant for the disease, with subsequent use of molecular biology methods to uncover the exact mechanisms. This review indicates the trend of combining speciation and neuroscience and provides a sketch about data and outcomes. For brain research, we recommend using modern, powerful techniques throughout which provide advanced validity and information in a chemical sense.

Introduction

Element speciation is an important field within analytical chemistry since already more than about thirty years. For definitions of speciation related terms the reader is referred to IUPAC regulations or the paper of Templeton et al. [1]. Speciation analysis offers deeper insight into molecular mechanisms and pathways of disease by determining the speciation of an element – the pattern of distinct element binding forms. Speciation research can compare such species pattern between healthy controls and patients for disease-related changes or shifts. Having such valuable potential for investigating mechanisms of neurodegenerative conditions, speciation research is increasingly considered in neurological studies within clinical or epidemiological context. Elemental speciation nowadays is providing an important bridge between powerful techniques of analytical chemistry and neurodevelopment or brain degeneration research [2]. Neurological disorders and age-related dementia are pressing problems in an aging society. Alzheimer's disease (AD) is the most common age-related disorder, affecting two percent of total population, and it is expected to increase above fifty percent in elderly over sixty-five years in the USA. Parkinson's disease (PD) and mild cognitive impairment (MCI) are further neurodegenerative disorders with strongly increasing prevalence [3]. The etiology of AD, PD, and MCI is closely linked to oxidative stress followed from cascades of protein misfolding and other metabolic changes. Aside from further reasons, oxidative stress, in turn, is also caused by misbalances of essential metals with redox capability, such as iron, copper, and manganese, or by exchange or exposure to adverse elements like aluminum, mercury or arsenic. Another neurodegenerative condition is amyotrophic lateral sclerosis (ALS), a motor neuron disease, whose etiology remains substantially unknown, except for few cases with gene mutations [4]. Environmental factors are, however, suspected causative. Epidemiological studies pointed to selenium as a risk factor for ALS, which however is somewhat contradictory to molecular biology studies based on knockout animal models, showing mainly that Se species are neuroprotective, especially SELENOP and GPX [5], [6].

Element speciation has matured to provide key-knowledge by investigating changes in both species concentration and pattern of essential elements (e.g. Mn–Tf vs. Mn–citrate; SELENOP vs. Se(IV)) or shifts in their redox pairs (e.g. Fe(II) vs. Fe(III)).

An important issue is samples and sample matrices for speciation studies. Due to simplicity in sample availability, blood and serum are still used in most studies. However, according to the selective permeability of NB, element species composition, species concentration and pattern in the brain or CSF – or more general speaking, beyond NB – are typically independent and may be completely different from cycling fluids in the body [7]. Apart from different information gained by imaging techniques like MRT, CSF offers the closest chemical analysis view on the brain, while it is operating in its actual physiological or diseased condition. CSF is an excretion of the choroid plexus and is in permanent close contact to brain in the extraparenchymal cave [8]. The use of CSF as specimen for speciation also circumvents the need for extrapolation to humans from results gained by animal models. Consequently, the sample type of choice from living humans is CSF. However, limitations also have to be considered: by law, drawing CSF is only allowed after a strict medical indication. The primary importance for sampling is the sake of the patient. Sampling procedures have to follow medical needs and standards (disinfection = danger of contamination), using stainless steel needles (=contact to Mn and Fe). Analytical demands stand behind such medical constraints.

Importantly, it should be stressed that for the case–control studies, involving CSF analysis, the control CSF samples originate from “neurological healthy persons”. This means, that such persons initially seemingly had unspecific neurological complaints, provoking CSF sampling, but subsequent clinical chemistry excluded neuronal adverse conditions. CSF from healthy controls without any complaints is unavailable according to ethical standards and legal regulations. Also, it must be considered that samples from CSF banks could show limited suitability for speciation as samples were initially intended for different use. Sampling or storage history may be unclear or even unsuitable for later speciation studies. Due to the above limitations, animal studies are inevitable. These are performed as a compliment to the CSF studies, specifically for speciation investigations after defined exposure to metals or other chemicals.

Speciation techniques in use for neurobiology research are in general the same as applied for other research fields. Such methods are summarized already in various reviews, including their suitability for different matrices or element species, and the reader is referred to those articles for the basic technical information [9]. Overall, techniques used in neurodegeneration studies, too, consist of hyphenated methods, mostly HPLC coupled to ICP–MS. For QC and species identification orthogonal 2D-approaches are employed, using e.g. CE as independent separation device. Complementary species information is partly achieved by ESI–MS/MS or ESI–ICR–MS. The overview of the general workflow for speciation analysis in neurobiology, with the QC stressed, is presented in Fig. 1.

A matter of concern is species- and redox-stability during extraction procedures from the brain (experimental animals or human post mortem studies) and/or during species separation of brain extracts or CSF samples. Covalently bound stable element species, like selenium in SeMet, tolerate reversed-phase or ion-exchange chromatography for species separation, whereas currently most interesting element species, regarding AD or PD, e.g. Mn–citrate, Mn–Tf, are typically at risk to be transformed during sample preparation and species separation. Therefore, during sample preparation low-temperature and inert-gas atmosphere are typically necessary and smooth separation techniques with often only limited chromatographic resolution can be used [10].

In molecular biology studies, which mostly are based on knockout animal models [11], [12], “single-species assays” commonly are applied to follow changes of one specific element species. Then, specific single species like SELENOP are determined after e.g. relevant genes were knocked out and the related metabolic pathways are changed [13]. In such approaches, the specificity of the assay is of paramount importance. The subsequent sections will indicate that analytical techniques of speciation research have started to be introduced into neurological research and during recent years this changed from a beginning initiative to an established application trend resulting in fruitful cooperation between neurologists, epidemiologists, molecular biologists, and analytical chemists.