Schell LS UROP Mn Poster Final
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This study measured manganese (Mn) levels in different regions of post-mortem human brain samples using instrumental neutron activation analysis. Mn concentrations were highest in the anterior putamen and lowest in the white matter. Higher Mn levels in the anterior putamen were correlated with lower scores on scales measuring Alzheimer's disease symptoms. The results provide support for future studies exploring relationships between Mn levels, neurodegeneration, and Alzheimer's disease risk.
![EPIDEMIOLOGY OF MANGANESE
Anatomical 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.
ABSTRACT
Manganese (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 Chemistry
College of Arts and Science
University of Missouri
RESEARCH FUNDING
This 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
Lance A. Schell1,2, John D. Brockman1, and J. David Robertson1, 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 DISCUSSION
Samples 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 WORK
Results 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)
METHOD
Brain 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
0
0.5
1
1.5
2
2.5
3
3.5
4
Anterior Putamen Lateral
Cerebellum
Mid-Frontal Inferior Temporal White Matter
MNCONCENTRATION(PPM)
REGION
NIA-R: 2 NIA-R: 4
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
0
2
4
6
8
10
12
830 835 840 845 850 855 860 865 870
ln(Counts)
Energy (keV)
30 sec 3 min
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)](https://image.slidesharecdn.com/3974dad0-47f8-457c-a8a6-5401e64f3dbe-150207230525-conversion-gate01/85/Schell-LS-UROP-Mn-Poster-Final-1-320.jpg)
Schell LS UROP Mn Poster Final
- 1. EPIDEMIOLOGY OF MANGANESE Anatomical 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. ABSTRACT Manganese (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 Chemistry College of Arts and Science University of Missouri RESEARCH FUNDING This 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 Lance A. Schell1,2, John D. Brockman1, and J. David Robertson1, 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 DISCUSSION Samples 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 WORK Results 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) METHOD Brain 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 0 0.5 1 1.5 2 2.5 3 3.5 4 Anterior Putamen Lateral Cerebellum Mid-Frontal Inferior Temporal White Matter MNCONCENTRATION(PPM) REGION NIA-R: 2 NIA-R: 4 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 0 2 4 6 8 10 12 830 835 840 845 850 855 860 865 870 ln(Counts) Energy (keV) 30 sec 3 min 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)