Pawan Kumar1, Sadhana Kumari1, Senthil S Kumaran1, Shefali Chaudhary1, Vinay Goyal2, Madhuri Behari2, S N Dwivedi3, Achal Srivastava2, and Naranamangalam R Jagannathan1
1Department of NMR and MRI Facility, All India Institute of Medical Sciences, New Delhi, India, 2Department of Neurology, All India Institute of Medical Sciences, New Delhi, India, 3Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India
Synopsis
We used 1H NMR technique to investigate
the serum samples of 6 patients with Parkinson’s disease (PD) and 6 healthy
controls (HC) using 700 MHz NMR spectrometer and the data were processed using
the Agilent software, Vnmrj2.3A. PLS-DA multivariate analysis was performed to
explore biochemical dissimilarities between PD patients and HC using
MetaboAnalyst (3.0) software. We observed significantly elevated levels of lactate,
glutamate and methyl guanidine PD patients in comparison with HC on t-test, suggesting
protein metabolism impairment, mitochondrial dysfunction and oxidative stress
in PD patients.Purpose
To evaluate the comprehensive metabolic profile of
serum in patients with PD.
Introduction
NMR spectroscopy of bio-fluids provides a wealth of
information about the metabolic processes of living organism
1. Parkinson’s
disease (PD) is a neurodegenerative disorder caused by selective loss of
dopaminergic neurons in the substantia nigrapars compacta which leads to
dysfunction of cerebral pathways critical for the control of movements. The
diagnosis of PD is based on motor symptoms, such as bradykinesia, akinesia,
muscular rigidity, postural instability, and resting tremor, which are evidently
after the degeneration of a significant number of dopaminergic neurons
2.
We investigated the metabolic profile differences in serum samples using
1H
NMR technique to differentiate between patients with PD and healthy controls,
and if possible, identify biomarkers for early diagnosis of PD.
Methodology
Blood samples were collected from 6 PD patients (4M/
2F, mean age: 57 ± 7.0 years) and 6 age and gender matched healthy controls (3M/3F,
mean age: 49 ± 2.22 years) after 12 hours fasting, centrifuged at 2000g for 10
min at 4 ͦC and stored at -80o C
until NMR experiments were performed. The diagnosis of PD was made according to
the UKPDS (United Kingdom Parkinson Disease Society) brain bank criteria3.
For NMR experiments 200µl serum and 30µl TSP (0.5mM) were added in D2O
making total volume to 600µl sample and NMR experiments were carried out using
700 MHz NMR spectrometer (M/s. Agilent Technologies, USA). The chemical shifts
of resonances referenced to TSP. Proton NMR spectra of serum samples were acquired
using 1D CPMG with presaturation using 90˚ pulse sequence, with 64 number of
scans; relaxation delay=70s; spectral width= 9000Hz; data points=32k; echo
time=15ms. 2D NMR Spectroscopy was carried out for assignment of resonances of 1D
spectra. The data is processed and integrated values were obtained using the Vnmrj2.3A software.
Statistical Analysis: PLS-DA
multivariate analysis was performed to explore biochemical dissimilarities
between PD patients and HC using MetaboAnalyst (3.0), a web-based metabolomics
data processing software.
Results
From the obtained spectrum from
blood sera (Figure 1), 15 metabolites were assigned unambiguously using 1D and
2D NMR and the integration of those metabolites were evaluated. For comparison between
PD and HC and estimation of significant difference, t- test (non-parametric
tests - Wilcoxon rank-sum test) was used. Significant increase in the integral
values of Lactate, Glutamine and Methyl Guanidine were observed in PD patients as
compared to healthy controls (Table 1).
PLS-DA analysis depicts clear separation between PD and HC (Figure 2).
Discussion and conclusion
The elevated Methyl Guanidine concentration in PD
as compared to healthy controls may be attributed to oxidative stress and
protein metabolism impairment
4. Increased levels of glutamate may be
ascribed to impaired mitochondrial function, due to increased vulnerability of
affected neurons
5. The above results suggest that NMR based
metabolomics study may be useful for understanding the pathogenesis of PD and
further help in identification and establishment of biomarker(s) for the
diagnosis of PD.
Acknowledgements
No acknowledgement found.References
1.
Lindon, JC, Nicholson JK, Holmes E, Everett JR.
"Metabonomics: metabolic processes studied by NMR spectroscopy of
biofluids." Concepts in Magnetic
Resonance 2000;12: 289-320.
2.
Ciurleo, R, Di Lorenzo G, Bramanti P, Marino S.
"Magnetic Resonance Spectroscopy: An In Vivo Molecular Imaging Biomarker
for Parkinson’s Disease?." BioMed
Research International 2014;2014;519816
3.
Martínez-Martín, P, et al.
"Unified Parkinson's disease rating scale characteristics and structure."
Movement Disorders 1994; 9: 76-83.
4.
Raquel D, et al.
"Oxidative stress and aminopeptidases in Parkinson’s disease patients with
and without treatment." Neurodegenerative Diseases 2011; 8: 109-116.
5.
Beal, MF, Lang
AE, and Ludolph AC. Neurodegenerative Diseases: Neurobiology, Pathogenesis and
Therapeutics. Cambridge University Press, 2005; ISBN:9780511113741.
5.
Beal, MF, Lang
AE, and Ludolph AC. Neurodegenerative Diseases: Neurobiology, Pathogenesis and
Therapeutics. Cambridge University Press, 2005; ISBN:9780511113741.