senior lecturer, department of biotechnology, tel-hai academic collage, israel & oxidative...
TRANSCRIPT
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Senior lecturer, Department of Biotechnology, Tel-Hai academic collage, Israel&
Oxidative Stress and Human Diseases Research Laboratory – Galilee Research Institute (Migal), Israel.
Analysis of volatile organic compounds in rats with dopaminergic lesion:
possible application for early detection of Parkinson’s disease
By
Dr. Soliman Khatib
Oxidative Stress laboratory
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Oxidative stress and human diseases laboratory activities
Markers
Alzheimer, Parkinson, diabetic complications,
atherosclerosis, depression
Analytical chemistry Isolation, Identification
Protein oxidation and modifications
Blood Human carotid plaque
N
NH
N
NH2
ON
OCH
2
O
OH
O
O
OH
CH2
NH
VOCs as Biomarkers
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Parkinson’s disease
• Parkinson's disease (PD): one of the most prevalent neurodegenerative disorders
• Affecting :• 0.3% of the entire population, • 1% of the people over 65yr, • 4% of the people over 80yr.
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Parkinson’s Disease (PD) Pathology
• Pathology is characterized with the death of dopaminergic neurons in the substantia nigra Para compacta and a reduction in dopamine (DA) levels in the striatum and the presence of “Lewy Bodies”.
NH2
HO
OH
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Symptoms of PD
• Tremor
• Rigidity
•Bradykinesia
• Postural instability
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loss of 50-70% of Dopaminrgic neurons
80% reduction in the striatal dopamine
Parkinson’s disease and dopamine
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Early detection of PD
• Early detection of PD is important as it enables slowing or stopping its progression using neuroprotective drugs.
• Although there are several biomarkers for clinical diagnosis, none of these is in routine clinical use.
• Thus, a need for an early, reliable, sensitive and specific diagnostic biomarker still exits.
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Aim of the study
Early detection of PD
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PD
9
Volatile organic compounds (VOCs) in blood
Early detection of PD stage and progression
Biochemical pathways specific to the disease
Hypothesis
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Objectives
• 1. To identify VOCs in rates blood that can be specific to Parkinson disease.
• To investigate the mechanism of such specific VOCs generation and their association with the disease.
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• VOCs can be detected by using the simple and reliable Solid-Phase Micro Extraction (SPME) technique, combined with Gas-Chromatography Mass Spectrometry (GC-MS).
SPME Method
0 5 10 15 20 25 30 350
1
2
3
4
5 x 106
5.5 6 6.5 7 7.5 8 8.5 9 9.5
0
1
2
x 105
5.5 6 6.5 7 7.5 8 8.5 9 9.5
02468
x 104
5.5 6 6.5 7 7.5 8 8.5 9 9.50
2
4
x 104
5.5 6 6.5 7 7.5 8 8.5 9 9.50
10
20x 10
4
5.5 6 6.5 7 7.5 8 8.5 9 9.505
1015
x 104
Retention time, [min.]
RT=9.004
RT=8.998
RT=9.004
RT=9.010
RT=9.004
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1. Fiber type: (DVB/CAR/PDMS) (gray fiber) or (PDMS) (red fiber).
Optimization of the SPME method
2. SPME Extraction Temperature ( 40C Vs 90C)
3. Blood or Serum
Gray fiber
Red fiber
400C
900C
Blood
Serum
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The requirement for the algorithm
• In each experiment – numerous GC-MS peaks• Each chromatogram contains hundreds of peaks• The need for an automatic and reliable algorithm
for data handling, signal processing and analysis
13
0 5 10 15 20 25 30 350
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5x 10
7
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Development of an algorithm for data handling and processing.
• accurately detecting and demarcating the peaks in the GC-MS chromatogram.
• calculating the area under each detected peak
• eliminating peaks originated from blanks,
• Normalization of the area under each peak using internal standards
• finding and selecting sample peaks with areas significantly higher or lower than in control.
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Baseline computation and removal
24.2 24.4 24.6 24.8 25 25.2 25.4 25.6 25.8 26 26.20
1
2
3
4
5
6
7
8x 10
4
Retention time (minutes)
15
26 27 28 29 30 31 32 33 34
1
1.5
2
2.5
3
3.5
4x 10
4
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• “Peak picking”• Selecting peaks:
peak-to-noise ratio>3dB
• Left and right boundaries
24.2 24.4 24.6 24.8 25 25.2 25.4 25.6 25.8 26 26.20
1
2
3
4
5
6
7
8x 10
4
Retention time (minutes)
16
Peak detection and boundary demarcation
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Peaks alignment and grouping
5.5 6 6.5 7 7.5 8 8.5 9 9.5
0
1
2
x 105
5.5 6 6.5 7 7.5 8 8.5 9 9.5
02468
x 104
5.5 6 6.5 7 7.5 8 8.5 9 9.50
2
4
x 104
5.5 6 6.5 7 7.5 8 8.5 9 9.50
10
20x 10
4
5.5 6 6.5 7 7.5 8 8.5 9 9.505
1015
x 104
Retention time, [min.]
RT=9.004
RT=8.998
RT=9.004
RT=9.010
RT=9.004
Aim: targeting peaks partial GC/MS signals from 5 different samples, each contains several local maxima, aligned on a common time-scale. The retention times of peaks which stem from the same compound are shown for all samples, demonstrating the small shifts between different samples.
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Area computation and Internal Standard Correction
Interpolation
Win= 5
Time Alignment & Clustering
k
Area Computation
Low-pass Filtering
win=50
Area Computation
Outlier Selection
Sample peaks
Baseline signal
1
N
2
Internal Standard Areas
Blank peaks
18
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Area versus retention-time of all peaks in all blood samples. Dotted red lines represent peaks originated from blanks, and solid blue lines represent peaks where no blanks were present.
Peaks vs. Blanks
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Rat model: Stable- 50% depletion of dopaminergic neurons by injection of 6-OHDA i.c.v into the left lateral ventricle (Aluf et al. FRR, 2010).
SHAM
125µg 6-OHDA
250 µg 6-OHDA
325 µg 6-OHDA
0.00
0.25
0.50
0.75
1.00
1.25
**
**
**
Sur
vivi
ng c
ells
Rat
io f
rom
con
trol
**)P<0.01, one way ANOVA, n = 7(
PD Rates model
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Method
1- Rates were injected by i.c.v into the left lateral ventricle with 250 g 6-OHDA, or i.p injection of (DSP-4), 50 mg/kg or seline.
2- after 5 weeks blood was collected.
3- rats were decapitated, the brain removed and the striatum dissected , weighted and homogenized into PBS.
4- dopamine content in striatum determined using HPLC connected to ECD.
5- VOCs from Blood and striatum homogenate were determined using SPME-GC-MS method PBS was used as blank.
6- data processed and analyzed using the developed algorithm.
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Dopamine levels in the striatum homogenate of rats
DA= Dopamine
HDA= 6-hydroxydopamine (HDA)
DSP-4= N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine
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The level of VOCs in treated versus control rats as analyzed by SPME/GC-MS.
A: Blood
B: Brain homogenate
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The MS fragmentations of peaks
spectra of 1-Octen-3-ol from blood
spectra of 1-Octen-3-ol from standard
spectra of 2-Ethylhexanol from blood
spectra of 2-Ethylhexanol from standard
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VOCs detected in blood or Brain Homogenate
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VOCs detected in blood or Brain Homogenate
1-Octen-3-ol is neurotoxic in the Drosophila melanogaster model and to human embryonic stem cells. (Inamdar et al Neurotox Res 2014)
it reduces dopamine levels, causes dopamine neuron degeneration. (Inamdar et al. PNAS, 2013;)
1-Octen-3-ol has been detected in cow milk (van Aardt et al., 2005),
human sweat (Cork and Park, 1996) , and feces (Garner et al., 2007).
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Summary
• Three groups of rats were studied: DA-lesioned rats injected with 6-hydroxydopamine ( n=11); control rats injected with saline (n=9); and control rats injected with DSP-4 (n=8), a specific noradrenergic neuron toxin.
• The SPME method was optimized for testing Blood VOCs
• An algorithm for data processing was developed
• In the blood, 1-octen-3-ol and 2-ethylhexanol were found at significantly higher concentrations in HDA-treated versus sham rats.
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Summary
• In the striatal homogenate 1-octen-3-ol and other four compounds were found at significantly lower concentrations in HDA rate
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Future plans
•To examine the presence of volatile materials in Parkinson human blood.
•To investigate the mechanism of the obtained VOCs generation and their association with the disease.
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III. Novel markers for extra cellular OS2. Extra cellular OS: Microdialysis of a non dialyzable marker
(ROS/RNS trap).
InletInlet
Outlet
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Oxidative stress- extra cellular marker oxidation
•Increased oxidative stress is observed in our rat model (44% and 28% increase in the level of oxidized products of the marker),•NAC decreased the increment almost back to normal
(n=5/6 rats per treatment; ***p<0.001, **p<0.01, *p<0.05)
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Thanks
• Prof. Jacob Vaya.
Dr. Soliman Khatib.
Saeed Mahmoud and our group.
• Prof. John Finberg
Dr. Yuval Aluf.
• Dr. Yizhar Lavner.