Chapter 20
Applications of Radioanalytical Chemistry to Alzheimer's Disease Downloaded by UNIV OF GUELPH LIBRARY on July 19, 2012 | http://pubs.acs.org Publication Date: November 4, 2003 | doi: 10.1021/bk-2004-0868.ch020
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J . D . Robertson and M. A. Lovell 1
Department of Chemistry and Missouri University Research Reactor, University of Missouri, Columbia, M O 65211 Sanders-Brown Center on Aging, University of Kentucky, Lexington, K Y 40536 Department of Chemistry, University of Kentucky, Lexington, K Y 40506 2
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Two examples of the use of radioanalytical methods to probe key questions about the pathogenesis o f Alzheimer's disease (AD) are presented. In die first, micro-beam proton-induced X-ray emission analysis was used to investigate imbalances of Zn in senile plaques (SP) and neurofibrillary tangles (NFT) i n A D in light o f observations that this metal can accelerate aggregation of amyloid beta peptide in vitro. In the second, accelerator mass spectrometry and the C bomb pulse were used to determine die average "age" of N F T and SP in the AD brain to help define the time course and defining factors involved in the formation of these pathological features. l4
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© 2004 American Chemical Society In Radioanalytical Methods in Interdisciplinary Research; Laue, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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299 Alzheimer's disease (AD), an age associated dementing disorder, is the most common form of adult onset dementia and is the fourth leading cause of death in the United States (/). A community-based study suggests that 4 million persons in the U S have A D (2) and that, with the aging of society, nearly 9 million individuals could be affected by the year 2040 (3). Clinically, A D is characterized by a gradually progressive decline in cognitive function and pathologically by neurofibrillary tangle (NFT), senile plaque (SP) and neuropil thread formation, and neuron and synapse loss, particularly in the hippocampus, amygdala, entorhinal cortex, neocortex and nucleus basalis of Meynert. The major histopathologic features of A D are senile plaques (SP), composed primarily of amyloid-β (AB) peptide and neurofibrillary tangles (NFT), composed of paired helical filaments containing hyperphosphorylated tau. These pathologic lesions markers remain the key criteria for the neuropathologic diagnosis of A D (4). The major barrier to treating and/or preventing A D is a lack of understanding of the etiology and pathogenesis of neuron degeneration. Numerous etiologic/pathogenic mechanisms have been suggested for the cause of A D including: genetic defects, metabolic defects, oxidative stress, endogenous toxins, mitochondrial defects, latent or slow virus, trace element toxicity or some combination of the above. In this work, we review our use of radiochemical methods to investigate the potential role of trace metals in A D and the time course involved in the formation of N F T and SP.
Trace Metals and AD A decade ago, the association of metals and A D conjured up thoughts of tossing out aluminum cookware and deodorant and not drinking beverages from aluminum cans. The hypothesis that exogenous aluminum (Al) plays a role in the development of A D now receives little attention, in part, because of radioanalytical studies of A l in A D brain. Ehmann and Markesbery used instrumental neutron activation analysis (BMAA), along with graphite furnace atomic absorption spectrometry (GFAAS) and laser microprobe mass spectrometry ( L M M S ) to study possible A l imbalances in brains of A D patients compared with age-matched controls. While slight elevations of A l were observed in small, bulk A D brain samples using G F A A S and in some A D brain cellular/subcellular components of the hippocampus using L M M S . INAA analyses did not detect an A l imbalance in A D brain when much larger samples were taken from a wider range of brain regions (5). Moreover, Watt and Grime et al. demonstrated through a series of micro-particle induced x-ray emission (μΡΙΧΕ) measurements that A l was not, as proposed, enhanced in SP and N F T of A D brain (6). While several other elements have been implicated in A D , including H g (7) and S i (5), Z n has become the focus of considerable interest. Zinc is a crucial component in over 300 enzymes including Cu/Zn superoxide dismutase, and in
In Radioanalytical Methods in Interdisciplinary Research; Laue, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
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various eukaryotic transcription factors. The possible connection of Z n to A D was first proposed by Burnett who suggested that Z n deficiencies lead to dementia and that Z n supplements might delay or prevent the onset of dementia The brain contains three Z n pools: 1) a membrane bound metalloprotein; 2) a vesicular pool localized in nerve terminal synaptic vesicles; and 3) an ionic pool of free or loosely bound ions in the cytoplasm (10). O f these sources, the vesicular pool, which is easily chelated, is thought to be the most important because it is released with glutamate during neurotransmission and may reach concentrations of 300 μΜ in the synapse. Unless these gradients of Z n are immediately sequestered they could potentially induce neurodegeneration. Although Z n is essential for normal fonction of the brain and may act in a protective role at low concentration, experimental studies have shown high (100 and 1000 μΜ) concentrations of Zn are toxic to neurons in vitro (11, 12) and in vivo (13,14). In 1994, Bush and co-workers reported that Z n at low physiological concentrations induced Α β aggregation in vitro (15,16). These reports, and the I N A A studies that showed significant Z n elevations at the "bulk" level in A D hippocampus and amygdala (7,17), led us to investigate the association between Zn and SP in A D brain using the radioanalytical technique micro-PIXE. The measurement is challenging because SP are only 20 to 40 microns in diameter and because Z n occurs at trace (10 to 100 μg/g) levels in the plaques and surrounding tissue. The technique of using an accelerated particle beam for x-ray emission analysis was first introduced at the Lund Institute o f Technology in 1970 (18). P I X E , like other elemental analysis x-ray spectroscopic techniques, utilizes the x-rays that are emitted from the atoms in a sample when that sample is exposed to an excitation source. The energies of the resultant x-rays are characteristic of the elements from which they are emitted and the number of x-rays of a given energy is proportional to the mass of that corresponding element in the sample. The use of a proton beam as an excitation source offers several advantages over other x-ray techniques including a higher rate of data accumulation across the entire periodic table and better overall sensitivities, especially for the lower atomic number elements. A s illustrated in Figure 1, the better sensitivity of P I X E in comparison to electron excitation is due to a lower bremsstrahlung background (see 19). The fractional mass sensitivities achieved with P I X E are typically on the order of 1 μ% per gram for samples whose total target masses range between 10 mg for standard systems to 1 pg for a proton microprobe. O f course, the chief disadvantage of P I X E is the reason that it is considered a radioanalytical technique; it requires the use of a particle accelerator. The results of the micro-PIXE analysis of ten SP and surrounding neuropil from 9 A D subjects and neuropil from 5 age-matched control subjects are
In Radioanalytical Methods in Interdisciplinary Research; Laue, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.
301 presented in Table I. A detailed description of the micro-PIXE measurements can be found in reference (20). Comparison of overall SP Z n levels with A D neuropil using the Mann-Whitney i/-test (non-normal distributions) demonstrated a significant elevation of Z n (p
