High Resolution 1H NMR-based Metabolomics study of Serum in Parkinson’s disease
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 organism1. 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 neurons2. 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 impairment4. Increased levels of glutamate may be ascribed to impaired mitochondrial function, due to increased vulnerability of affected neurons5. 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.

Figures

Figure1. Representative one dimensional 1H NMR spectrum of serum samples at 25oC in D2O of a PD patient (red) and a HC (black) on same scale.

Figure 2. PLS-DA scores plot of 1H NMR spectra of serum sample of PD (red) and healthy controls (green), in 2D (figure 2A) and 3D representation (figure 2B)

Table 1. Metabolites that were significantly different on t-test between Parkinson’s disease (n=6) and healthy controls (n=6)



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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