Introduction

Parkinson’s disease (PD) is a chronic neurodegenerative condition characterized by motor symptoms as a result of dopaminergic degeneration in the substantia nigra pars compacta¹. It affects 1% of the population above 60 years of all races and culture². Diagnosis of Parkinson's disease is established after the manifestation of motor symptoms like rigidity, bradykinesia, and tremor. However, recent studies support the hypothesis that nonmotor symptoms, particularly gastrointestinal dysfunctions, might serve as early biomarkers that lead to malnutrition³. As per Braak's hypothesis, the disease is assumed to originate in the intestine and via the vagus nerve it spreads to the brain⁴. Therefore, it has been hypothesized that peripheral dopaminergic impairments would possibly precede the alteration of dopaminergic neurons within the central nervous system and, finally, the appearance of motor symptoms. Thus, considering the complex process of diagnosis, there is a need for additional non-invasive biomarkers for the diagnosis and prognosis of PD. The aim of our study was to estimate how different stages of PD and dopaminergic treatment taken by the patients may affect the metabolic profile in the saliva of PD patients.

Methods

The study included early PD group (patients with early stages of the disease; H&Y stage≤ 2; n=52; mean age 53.5±7.3 years) and advanced PD group (patients with advanced stages of the disease; H&Y stage > 2; n=24; mean age ±SD: 57.9±8.2 years) and HC (n=37; mean age±SD=53.0±8.53 years) after the ethical approval (Table 1). The participants were instructed to refrain from any medication, food, smoke, drink or use of oral hygiene products for at least 12 hrs prior to saliva collection. Saliva samples were collected in the morning (between 9:00 and 11:00 am) to avoid the diurnal variations and were immediately stored at -80˚C. For the NMR experiments, saliva samples were thawed at room temperature and centrifuged at 2000g for 10 min at 4^oC to remove insoluble materials, food remnants and cell debris. 400µl of saliva sample was mixed with 170µL phosphate buffer solution (0.5 mM Na₂HPO₄/ NaH₂PO₄, pH 7.4, 99.9% D₂O) to maintain the pH variation across samples. Further, 0.5 mM TSP (3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt) was added to the sample for serving as an internal standard for concentration estimation as well as chemical shift reference. A total of 600 μl obtained solution were transferred into 5 mm tubes for the NMR experiments. Proton NMR spectra were acquired using 700 MHz NMR spectrometer (M/s. Agilent Technologies, USA) with 1D PRESAT using a 90˚ pulse, 128 scans; relaxation delay=14s. Assignments of various metabolite resonances were made using 1D & 2D NMR spectra. Data were integrated using Vnmrj software (version 2.3, Agilent) and further concentration was estimated. Significant metabolites were estimated by Mann-Whitney U test using STATA software (version 12).

Results and discussion

The metabolic profiles of 113 saliva samples were obtained from healthy and PD subjects. The assignments of a total of 40 salivary metabolites were carried out unambiguously (Figure 1). The observed metabolites include organic acids, neurotransmitters, short-chain fatty acids, amino acids, amines, and alcohols. Patients with early PD exhibited higher concentrations of histidine, TMAO, propionate, GABA, valine, isoleucine, alanine, and fucose in comparison with HC, indicating the characteristic changes of metabolite levels during the onset of PD, though it may be related to dopaminergic treatment also. Higher propionate and acetoin concentrations were observed in both early and advanced PD groups as compared to the HC group, suggesting these metabolites may not be altered by dopaminergic treatment. There were no differences between ePD and aPD groups (Figure 2). The concentrations of propionate (r=0.29, p=0.04) and acetoin (r=0.26, p=0.03) correlated with disease duration, but not with LEDD, H&Y stage and UPDRS (Figure 3). Elevated propionate and acetoin in patients with ePD and aPD and their positive correlation with disease duration suggests an association of PD biochemistry with the gut microbiome. Acetate and propionate are intestinal microbial metabolites that influence the formation of gut microbiota and the host metabolome⁵. Due to altered levels of these metabolites, intestinal microflora becomes imbalanced and may activate intestinal immunity to maintain the integrity of the gut barrier⁶. These findings suggest a possible relationship in oral health and bacterial status associated with the deterioration of periodontal health in patients with PD⁷. The concentrations of glycine (r=0.43, p≤0.01), taurine (r=0.52, p≤0.001), TMAO (r=0.36, p=0.01), isoleucine (r=0.30, p=0.04), and valine (r=0.31, p=0.04) correlated with LEDD suggest their association with dopaminergic treatment of PD patients. The concentration of butyrate (r=-0.25, p=0.04) metabolite had a negative correlation with H&Y stage and may be ascribed to disease severity, though it could not distinguish ePD and aPD. An increased concentration of metabolites in PD in comparison with HC may be attributed to a slower rate of amino acid absorption leading to malnutrition in patients with PD⁸.

Conclusion

The metabolites related to the gut microbiome, ketone body metabolism, and energy metabolism were affected in PD, with respect to healthy controls. However, the metabolites were not significantly different between ePD and aPD.

Acknowledgements

SK Acknowledges fellowship from CSIR.