EPIDEMIOLOGY OF MANGANESEAnatomical differences in the frontal cortex, cerebellum, basal ganglia, and white matter have been implicated in ADHD and several other neurological disorders, including AD. [1, 2] Diminished cognitive function, IQ, verbal ability, and ADHD-type symptoms have also been previously linked with high blood concentrations of Mn. [3] Current hypotheses suggest overexposure to Mn results in loss of total neuronal volume through α-synuclein oligomerization. [4] Increased oxidative damage due to loss of Mn-SOD and its cytoprotective effects could also help to explain this observation.

ABSTRACTManganese (Mn) is a trace element essential to the body’s natural processing of cholesterol, management of superoxide radicals generated during mitochondrial oxidative phosphorylation, and in overall growth and development. Human brain autopsy samples previously scored by a neuropathologist on the NIA Reagan Cross-Sectional and CERAD scales for likelihood of symptoms due to dementia or Alzheimer’s disease (AD) were analyzed using instrumental neutron activation analysis. Samples were irradiated for three minutes in high-purity quartz vials and decayed for one hour prior to counting on a high-purity germanium detector. Measured Mn concentrations in NIST Bovine Liver SRM 1577 material were 10.4 ± 0.4 µg/g. No observable detector or ambient Mn was encountered. Average Mn tissue concentrations in the anterior putamen, cerebellum, inferior temporal, mid-frontal, and white matter regions were 2.34 µg/g, 2.01 µg/g, 1.24 µg/g, 1.08 µg/g, and 0.79 µg/g, respectively. Limits of detection ranged from 0.06 µg/g for the white matter tissue to 0.16 µg/g for the inferior temporal tissue.

 

Anterior putamen Mn concentrations were inversely correlated with higher scores on both the NIA Reagan scale (p = 0.008) and the CERAD scale (p = 0.013). Previous reports have linked changes in the size of the putamen to the cognitive decline seen in patients with AD. Diminished Mn concentrations within this region might lead to loss of the anti-apoptotic properties associated with Mn superoxide dismutase, leading to frequent programmed cell death and less total neuron volume. Future epidemiological bioassays might target quantification of Mn levels as part of exploring SNPs in the superoxide dismutase enzymes within this region and their relationship to the prevention of neuronal apoptosis.

Department of ChemistryCollege of Arts and ScienceUniversity of Missouri

RESEARCH FUNDINGThis project was funded by an undergraduate research grant, awarded March 2013, through the University of Missouri Life Sciences Undergraduate Research Opportunity Program for academic year 2013 – 2014 and was also completed through the chemistry departmental honors course sequence.

ACKNOWLEDGEMENTS• Vicki Spate for irradiation and laboratory assistance• Ruth-Ann Ngwenyama for general laboratory support• Stacy Crane for previous sample preparation and data• Dr. Martha Claire Morris and Rush Medical College for

providing access to samples

Measurement of Manganese in Human Brain Autopsy Samples using Neutron Activation Analysis for Neuropathology Assessment in Alzheimer’sLance A. Schell 1,2, John D. Brockman 1, and J. David Robertson 1, 2

ranging from 30 seconds up to 3 minutes were tested, with the latter chosen because of a higher signal-to-noise ratio (Figure 5), while dead times remained under 20%. The limits of detection associated with the 3 minute irradiations were also significantly improved over lesser irradiation times for a given series of samples, as seen in Table 1.

Experimental Summary

• Liquid ICP-MS standards were centrifuged and freeze-dried.• Standards, SRM materials, and samples were encapsulated in

high-purity Haureus quartz with an open flame torch.• Samples were irradiated for 2 minutes with a geometry of 13

(1 SRM, 2 standards, 10 samples) in each irradiation carrier.• Following a one hour decay, gamma emissions from irradiated

samples were counted for ten minutes on a high-purity germanium (HPGe) semiconductor detector (Figure 4).

• Gaussian curves were fit to data using peak-fitting software.

RESULTS AND DISCUSSIONSamples from 10 patients in each of five brain regions were measured for levels of Mn. Levels were significantly different between each of the brain tissue regions. The results of this pilot study were promising – a two sided student’s t-test revealed decreased Mn levels as highly significant in the anterior putamen tissues (p<0.05). Figure 6 summarizes regional Mn concentrations found in this study between NIA-R scores of 2 and 4. Regression analysis on the anterior putamen data produced significant negative correlation coefficients on both the NIA-R (p = 0.008) and CERAD (p = 0.013) scales, indicating an inverse relationship.

Table 1. Irradiation Times and Limits of Detection (ng/g)

Region 30 Seconds 3 Minutes

Anterior Putamen 320 150

Inferior Cerebellum 290 130

Mid-Frontal 570 80

Inferior Temporal 430 150

White Matter 110 60

NIST 1577 Bovine Liver 90 10

NEUTRON ACTIVATION ANALYSIS

1 2

Figure 1. Human Brain Anatomy

FUTURE WORKResults from this study will be used as pilot data in a future NIH grant for collaboration with Rush Medical College to quantify Mn in several hundred brain tissue samples. Measured Mn concentrations will be used to assess the relationship of Mn with several neuropathologies, including Lewy bodies, cerebral infarcts, neural reserve, and the AD-specific amyloid depositions and neurofibrillary tangles, as well as several epidemiological cofactors (e.g. age, sex, education, and APOE-ε4).

OBJECTIVES• Development of an instrumental neutron activation analysis

(INAA) method for measurement of Mn in human brain autopsy samples encapsulated in high-purity quartz vials

• Measurement of Mn concentrations in brain samples from the anterior putamen, lateral cerebellum, mid-frontal lobe, inferior temporal lobe, and white matter in pilot samples.

• Comparison of measured concentrations with symptomatic assessment scales for Alzheimer’s Disease (AD)

METHODBrain autopsy samples were available from a previous NIH study focused on the potential role of Hg and Se in AD. Degree of AD symptoms was previously quantified on the NIA Reagan (NIA-R) and CERAD scales by neuropathologists at Rush Medical College.

Samples were analyzed using instrumental neutron activation analysis. 55Mn captures a thermal neutron and eventually emits a beta particle and gamma rays (Figure 2), becoming stable 56Fe. Irradiation was done using a pneumatic tube system at the University of Missouri with carrier vehicles shown in Figure 3. One of the emitted gamma rays at 846.8 keV was used for quantification. Mn concentrations measured in NIST 1577 Bovine Liver SRM material were 10.41 ± 0.41 µg/g, which compares well with the certified concentration range of 10.80 ± 1 ppm. Irradiation times s

Anterior PutamenLateral Cerebellum Mid-Frontal Inferior Temporal White Matter0

0.5

1

1.5

2

2.5

3

3.5

NIA-R: 2 NIA-R: 4

Region

Mn

Conc

entr

ation

(ppm

)

Figure 6. Regional Brain Mn Determinations

Figure 4. Autosampler

Samples in Queue

Detector (Lift)

Figure 3. Irradiations

Capped Rabbits

Polystyrene Quartz

x13

Figure 2. Overview of 55Mn(n,βγ)56Fe Reaction in NAA

55Mn

Thermal NeutronFlux

CompoundNucleus

56Fe

RadioactiveNucleusPrompt

Gamma

DelayedGamma

ß-

n 56Mn

830 835 840 845 850 855 860 865 8700

2

4

6

8

10

12

30 sec 3 minEnergy (keV)

ln (C

ount

s)

Figure 5. Optimization of Irradiation Time

CONCLUSIONS• A sensitive INAA method was developed for measurement of

Mn in human brain autopsy samples. Limits of detection were on the order of parts-per-billion in all samples and SRMs.

• Measured concentrations in the five regions were significantly different from each other.

• Anterior putamen Mn concentrations were inversely correlated with higher NIA Reagan and CERAD scores.

BIBLIOGRAPHY

1 Krain, A.L.; Castellanos, F.X. Clinical Psychology Review 2006, 26, 433.2 De Jong, L.W.; van der Hiele, K.; Veer, I.M., Houwing, J.J.; Westendorp, R.G.J.;

Bollen, E.L.E.M.; de Bruin, P.W.; Middelkoop, H.A.M.; van Buchem, M.A.; van der Grond, J. Brain 2008, 131, 3277.

3 Bouchard, M.; Laforest, F.; Vandelac, L.; Bellinger, D.; Mergler, D. Environmental Health Perspectives 2007, 115, 122.

4 Xu, B. Wu, S-W, Lu, C-W; Deng, Y.; Liu, W.; Wei, Y-G.; Yang, T-Y.; Xu, Z-F. Toxicology 2013, 305, 71.

(Right Brain Image Credit: Benjamin Cummings)