Parkinson’s disease is a complex, heterogeneous neurodegenerative disorder with an expected risen prevalence up to 9 million in 2030 [27]. Thus, an increasing number of PD patients might cause a high financial and social burden [28]. Clinical diagnosis of PD is usually established when first parkinsonian symptoms appear and when there is already a significant dopaminergic loss [9]. Therefore, due to the lack of biomarkers for early diagnosis of PD, targeting metabolites that could be involved in PD development and progression might improve therapeutic efficiency and provide a better understanding of the underlying molecular mechanisms that lead to PD development [27], as well as improving the quality life of patients and relieve pressure on medical services.
3.2. The altered metabolites
Out of the 40 studied compounds related to PD, seven statistically significant (p < 0.05) metabolites have been observed in PD subjects compared with healthy control subjects. Benzoic acid, palmitic acid, oleic acid, stearic acid, myo-inositol, sorbitol and quinolinic acid were significantly changed in PD subjects.
Altered levels of sugar alcohols, galactitol and sorbitol, might indicate alterations in the sugar metabolism, especially in galactose metabolism. It is assumed that increased glucose levels could overcome glycolysis capacity, which cause conversion of glucose to sorbitol. Another altered metabolite that was significantly altered is myo-inositol, which also plays an important role in sugar metabolism. It is known that altered levels of myo-inositol, together with altered levels of sorbitol might be associated with changes in polyols metabolic pathways, including glucose metabolism and glycolysis [8]. Alterations of glycolysis and sugar metabolism imply on potential involvement of metabolic pathways that participate in the energy production in pathogenesis of PD [10]. Besides, malabsorption of sorbitol might be associated with gastrointestinal dysfunction. Bacterial overgrowth causes changes in gut mucosa, what leads to sugar malabsorption [29]. Bacterial overgrowth, as well as other gastrointestinal dysfunctions are typical among subjects with PD. Up to 80% of PD patients show some signs of gastrointestinal impairments, which usually appear in the early stage of PD [30]. Altered levels of galactitol and sorbitol in subjects with PD have been observed in other studies too [10]. While we obtained decreased levels of galactitol and sorbitol, Ahmad and colleagues (2009) showed decreased levels of galactitol but increased levels of sorbitol [8]. However, even inconsistent results indicate possible association of PD pathogenesis with dysfunction of sugar metabolism and energy production and therefore represent potential biomarkers for early diagnosis of PD. While we found decreased levels of myo-inositol, Ahmad and colleagues (2009) found its levels increased in subjects with Parkinson [8]. Such changes indicate possible involvement of mitochondrial dysfunction in altered energy metabolism in the early stage of Parkinson’s disease or even before the illness has appeared.
Decreased levels of fatty acids in subjects with PD have been observed in this study. Palmitic, oleic and stearic acids were significantly decreased. Such a result is in correspondence with other studies that found reduction in fatty acids levels among PD patients [4]. Havelund et al. (2017) found, among other, decreased levels of palmitic, oleic and stearic acid in subjects with PD [4]. The metabolism of fatty acids has repeatedly been associated to the development and pathogenesis of PD. Changes in fatty acids might be associated with mitochondrial dysfunction [4, 5, 31], neuroinflammation [13], alterations in apoptotic signaling [32], as well as with oxidative stress [13]. Oxidative stress cause production of reactive oxygen species that affect fatty acids, making them more vulnerable for lipid peroxidation and causing membrane impairments, as well as loss of integrity [17]. Therefore, these changes in the metabolism of fatty acid could help to understand the progression of PD and the involved species could be potential biomarkers for early diagnosis of the disease.
In this study reduced levels of propylene glycol has been observed, while Ahmad et al. (2009) found propylene glycol levels in PD statistically elevated [8]. Propylene glycol is part of several metabolic pathways including glycine, serine, tyrosine and pyruvate metabolism, which dysregulation are associated to Parkinson’s disease [2, 3]. These metabolic pathways are related to dopamine and energy metabolism. It is known that the dysregulation of dopamine metabolism is characteristic for PD, including dopaminergic loss [4, 5]. Through mitochondrial dysfunction and dysregulation of energy metabolism, TCA cycle is associated with dopamine metabolism, while pyruvate is one of the metabolites that plays an important role in the TCA cycle. Therefore, altered levels of propylene glycol and its implication in several metabolic pathways, such as pyruvate, glycine metabolism, which are mutually interconnected, might indicate that the energy and dopamine metabolism are disrupted in the early stage of PD. Another altered metabolite indicates involvement of TCA cycle in PD pathogenesis. In this study, increased levels of succinic acid have been observed in PD subjects. Succinic acid is a part of TCA cycle, which is assumed to be downregulated in the early phases of PD [5]. These alterations of the TCA cycle might be result of mitochondrial dysfunction, as well as impairments in energy production. However, due to some inconsistent results, further research of potential implication of this metabolic pathway is necessary.
Quinolinic acid is an intermediate compound in the tryptophan-kynurenine metabolic pathway [7]. Kynurenine is the main intermediate compound and it can be metabolized in two ways by two kynurenine aminotransferase isoenzymes to kynurenic acid, which acts as neuroprotective agent, or to 3-hydroxykynurenine, and quinolinic acid, which are neurotoxic. Increased levels of quinolinic acid cause neuron excitation by activation of NMDA receptors, which consequently leads to excitotoxicity, increased inflammation and eventually to the neuronal death [33]. It is known that degeneration of dopaminergic neurons in the substantia nigra in Parkinson’s disease is a result of excitotoxcicity. Recent studies [6, 34] showed that altered kynurenine pathways and its metabolites are present in plasma and cerebrospinal fluid respectively in subjects with PD. This is in correspondence with our finding of altered levels of quinolinic acid. Dysfunction of tryptophan and kynurenine pathway might result in increased oxidative stress, as well as neuroinflammation that would lead to neurodegenerative processes characteristic for PD. Therefore, increased levels of quinolinic acid in the subjects that later developed PD might indicate alterations in the kynurenine pathway in the early stage, before first PD symptoms appear and might represent potential biomarker for early diagnosis or future improved treatment, that could target kynurenine to 3-hydroxykynurenine conversion [7].
Globally, the metabolites that have been found to be significantly altered in this study are related to mitochondrial dysfunction, the oxidative stress and the mechanisms of energy production. Considering that all the volunteers enrolled in this study were healthy at the time of sample collection, these finding might imply that these processes begin to be affected before PD shows any symptoms. This information is of great relevance for a disease such as PD, in which the definition of a metabolite panel that can be used for the early diagnosis of the disease has been sought for decades. Counting with the necessary tools for defining and studying such panels enables effective clinical managements, ensures early treatments and reduces the chances of complications.
3.3. EPIC samples for finding biomarker for the early diagnosis of PD
We are aware of the limitations of the study, in particular, in terms of the sample size. Therefore, these metabolites should be validated in larger studies. However, what we consider the most important aspect of the present work, is the use of true reliable samples that allow for well-founded biomarkers for the early diagnosis of PD. The samples used in this work are of invaluable importance. They were obtained from the large prospective EPIC study, in which volunteers were followed up for years, not just for cancer events, which was the main objective of the study, but for the development of many other chronic diseases such as cardiovascular disease, type 2 diabetes, PD and also mortality or even healthy ageing. We want to highlight that the samples included in this study, which include plasma, serum, leukocytes, and erythrocytes, are searchable and researchers can apply for their collection and use in their research studies. This work used samples from the EPIC cohort in Navarra (Spain). Among the 8084 participants enrolled in this cohort (Fig. 1), 36 were found to be suitable for our study. Two other EPIC-Spain cohorts have undergone the ascertainment of PD cases, for a total of 25,016 participants and 69 PD cases occurred during over 15 years of follow-up. However, Spain has a total of 5 cohorts, counting up to 41438 participants, who donated the different sample types mentioned before. And ours is just one of the 10 participating countries. All this, considered together, provides multiple options worth of investigation. In fact, the EPIC study can be branched in endless ways, which keep the project well alive.
One of the premises of this work was that some of the metabolites that are altered in conditions of established PD might also be altered before and, therefore, could be used as biomarkers for the early diagnosis of PD. In order to study such a premise, reliable samples must be used. In these regards, the samples from the EPIC study were a great opportunity for the evaluation of panels of metabolites that might be altered in subjects before they developed a disease. Being aware of the small cohort studied in this work, we have also limited our study to certain compounds that were found altered in previous studies. This does not exclude the possibility that other metabolic pathways could be altered before the disease is stablished and other studies of different natures should be performed, such as untargeted studies, which aims for the blind discovery of differential metabolites.