The present study is focused to identify the metabolic marker for the detection of PD from plasma and to validate the variations by spotting out through gene responsibility.
Several studies have been indicated the gene variation in PD [24
] which may lead to metabolic abnormalities that are detectable in peripheral tissues. Few successful break through have shown metabolite variations in CSF and serum samples of PD. For instance, glutamate was found to be decreased in parkinsonian CSF compared to control subjects, a result reported in earliest analyses of parkinsonian CSF [29
]. Subsequent study shows that the increased level of glycine, aspartate and glutamate in the plasma of parkinsonian patients [32
]. Increase in CSF glycine also been observed in PD [33
]. Previous study of PD patients showed reduction in concentrations of arginine and methionine in serum, while the level of valine was increased [34
]. Additionally, a significant decrease in CSF isoleucine, alanine, lysine and moderate increase of a glutamine level was very well observed in PD [34
]. Recent study shows the feasibility of potential diagnosis of PD from the detection of increased 8-OHdG in serum and urine of PD [35
]. Furthermore, several reports indicate that there is a reduction of complex 1 activity in the electron transport chain of PD [36
]. From the detailed knowledge of these studies, 22 metabolites, which play a role in mitochondrial function and other related pathway, have been targeted.
Analysis of the 1H NMR spectra of plasma samples showed differential distribution of these metabolites in drug-naive patients compared to the healthy volunteers. The targeted metabolite profiling of 22 compounds in blood plasma was characteristically altered in patients with PD, and most of these metabolites have decreased in concentration. The results showed that, this approach has great hope in diagnosis of PD. Moreover, this study was made with unmedicated PD patients to controls, to avoid the confounding effects of any medications. The key metabolites, such as myoinositol, sorbitol, citrate, acetate, succinate and pyruvate are significant in contribution for the separation between metabolite profiles of unmedicated PD patients and controls.
Plasma myoinositol level was significantly increased in the drug-naive patients. Elevation of myoinositol concentrations implies the decrease in activity of sciatic motor-nerve conduction velocity determined in animal models [42
]. Elevated plasma myoinositol has not previously been reported for PD. However, studies show the abnormal myoinositol levels in the brain of neurologically diseased patients and other disorders [44
]. Raised myoinositol levels in the basal ganglia were recently observed in PD patients under exercise condition detected using magnetic resonance spectroscopy [48
]. Additionally, plasma sorbitol level was significantly increased in drug-naive patients. Sorbitol has been linked to drug treatment in PD [49
], yet our observation of an elevation of plasma sorbitol concentrations in drug-naive patients may be due to impairment in oxidative stress [50
]. The elevated levels of sorbitol have been reported in the CSF of mood disorder patients, which relates to oxidative stress [52
]. Surprisingly, elevation in the plasma sorbitol level has not been reported in PD to our knowledge. Together with the significant findings of myoinositol and sorbitol in plasma imply the dysfunction of polyols metabolic pathway. Polyols pathway is a minor metabolic pathway of glucose running parallel to glycolysis, whose activity is altered in mitochondrial dysfunction [53
]. However, malfunctioning of mitochondria is previously reported in PD [36
], and it is the pedestal of this study. Detection of glucose concentration were not been carried out but abnormality of glucose metabolism was previously reported in PD patients [54
More interestingly citrate, malate, acetate, succinate and pyruvate are significantly varied in PD plasma samples, contributes to the major distinction of PD from normal samples in PLS-DA analysis. These metabolites, such as citrate, acetate, succinate and malate were decreased, while increase in pyruvate concentration was noticed. Pyruvate is the end metabolite of glycolysis. It enters Kreb's cycle as acetyl-coA by the catalysis of enzyme pyruvate dehydrogenase in the presence of the coenzyme NAD+. The accumulation of pyruvate or its increased concentration in plasma may be due to abnormal activity of pyruvate dehydrogenase complex and its interacting genes in patients. Increased pyruvate CSF has already been reported in Alzheimer's patients [55
]. The other intermediates of Kreb's cycle such as citrate, malate and succinate were considerably decreased in this study, which may correlate to alteration of pyruvate dehydrogenase activity. Moreover, the detection of other metabolites of Kreb's cycle was not executed in our analysis. Systems biological approach was carried out on pyruvate dehydrogenase components to identify its interacting genes which are hypothesized as the cause for the increased plasma pyruvate concentration. The analysis reveals 46 interacting genes together with pyruvate dehydrogenase components.
The significant variation in plasma pyruvate was validated by gene expression analysis of 46 genes derived from systems biological approach. The expression analysis was executed only on 40 genes and the other 6 genes in which the following FLJ21936, DMRT3, AKR1CL2, UNC5A and UNC5B were excluded from this study, because these genes are not expressed in blood cells and GCSL gene was also eliminated since it is considered as an alias of DLD gene. The analysis of 40 genes shows differential regulation of 16 genes in comparison to control. Interestingly, out of 16 genes, 9 genes have been previously reported in PD, such as CAT, FGF13, JUN, INSR, NOS1, OGDH, SYT1, FGF2 and SST [57
]. Furthermore, statistical analysis of these genes show the significance of NPFF and PDHB with p < 0.05. NPFF gene plays a major role in inflammation modulation, neuroendocrine function and cardiovascular regulations [66
], where as, PDHB gene is the beta subunit of pyruvate dehydrogenase, directly associated with pyruvate dehydrogenase activity and mitochondrial dysfunction. Abnormal activity of pyruvate dehydrogenase is the basis of our study in representing the variation in pyruvate concentration. The impairment of cardiovascular regulation [67
], inflammation [68
] and neuroendocrine function [69
] were reported in PD and this may suggest the influence of differential regulation of NPFF gene. The biological significance of these genes related to PD has not been reported previously. Hence, the variation in pyruvate concentration can be considered as a marker for detection of PD and future studies need to be carried out on the factors and the hidden mechanism involved in variations of NPFF and PDHB in PD.
Prior study indicates no correlation between metabolite variation with severity and duration in PD [24
]. To bring out the correlation, NMR peak amplitude representing the ~290 metabolite was included as one of the variables in the prediction of disease stages by neural network. The ANN was trained with variable parameters as described above. More fascinating results emerged from neural network and it is capable to predict early stages of PD, with a good accuracy using these variables. The optimized network yield has an accuracy of 97.14% in detecting PD patients. Based on the present study it has been confirmed that the association between disease progression and metabolite variation is strong and it is more accurate than the current diagnosis [23
] of PD.