Prognostic biomarkers of Parkinson’s disease in the Spanish EPIC cohort: a multiplatform metabolomics approach

The lack of knowledge about the onset and progression of Parkinson’s disease (PD) hampers its early diagnosis and treatment. Metabolomics might shed light on the PD imprint seeking a broader view of the biochemical remodeling induced by this disease in an early and pre-symptomatic stage and unveiling potential biomarkers. To achieve this goal, we took advantage of the great potential of the European Prospective Study on Nutrition and Cancer (EPIC) cohort to apply metabolomics searching for early diagnostic PD markers. This cohort consisted of healthy volunteers that were followed for around 15 years until June 2011 to ascertain incident PD. For this untargeted metabolomics-based study, baseline preclinical plasma samples of 39 randomly selected individuals that developed PD (Pre-PD group) and the corresponding control group were analyzed using a multiplatform approach. Data were statistically analyzed and exposed alterations in 33 metabolites levels, including significantly lower levels of free fatty acids (FFAs) in the preclinical samples from PD subjects. These results were then validated by adopting a targeted HPLC-QqQ-MS approach. After integrating all the metabolites affected, our finding revealed alterations in FFAs metabolism, mitochondrial dysfunction, oxidative stress, and gut–brain axis dysregulation long before the development of PD hallmarks. Although the biological purpose of these events is still unknown, the remodeled metabolic pathways highlighted in this work might be considered worthy prognostic biomarkers of early prodromal PD. The findings revealed by this work are of inestimable value since this is the first study conducted with samples collected many years before the disease development.


MS and LC-QqQ-MS) analyses
The 78 randomly selected plasma samples (pre-PD group, n = 39; control group, n = 39; gender balanced) were thawed on ice for approximately 1 hour. The samples were vortex-mixed for 2 minutes and 100 µL of plasma were transferred to an Eppendorf tube. Subsequently, 300 µL of a previously prepared cold mixture (-20°C) of methanol:ethanol (1:1, v/v) were added in the Eppendorf tube for deproteinization. After stirring the samples for 1 minute, they were incubated on ice for 5 minutes and vortex-mixed for another minute. Samples were centrifuged for 20 minutes at 13,000 rpm at 4 °C. After centrifugation, 100 µL of the supernatant was transferred to a chromatography vial with insert and was directly injected into the system.

Metabolites extraction for Gas chromatography coupled with mass spectrometry analysis
Once the samples were thawed on ice for 1 hour, they were vortex-mixed for 2 minutes and 40 µL of plasma were transferred to an Eppendorf tube. A volume of 120 µL of cold acetonitrile (-20 °C) was used for deproteinization. Sample preparation continued with vortex mixing for 2 minutes, incubation on ice and centrifugation for 10 minutes at 15,400 rpm at 4⁰C. Then, 100 µL of the supernatant were transferred to a GC-MS vial to be evaporated to dryness in a vacuum concentrator. Once the vials were completely dry, the derivatization process was continued to obtain volatile derivatives for analysis. First, 10 µL of O-methoxyamine in pyridine (15mg/mL) were added to each of the vials. The samples were then vortex-mixed vigorously for 5 minutes, and then 3 sonication cycles (2 minutes) and 3 vortex cycles (2 minutes) were performed. The samples were covered with aluminum foil and incubated at room temperature, in the dark to complete the methoximation process. After 16 hours of incubation, 10 µL of N,O 3 bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were added to all the samples, followed by 5 minutes of vortex-mixing. Samples were placed in the oven at 70 °C for 1 hour to complete the silylation reaction, which was followed by 30 minutes of cooling down. Finally, 100 µL of heptane with 20 ppm of tricosane (internal standard) were added to the samples and mixed for 1 minute in the vortex mixer.

Metabolites extraction for Capillary electrophoresis coupled with mass spectrometry analysis
After thawing the samples on ice for 1 hour, they were vortex-mixed for 1 minute and 100 µL of plasma were transferred to an Eppendorf tube. Subsequently, 100 µL of 0.2 M formic acid containing 5% acetonitrile and 0.4 mM methionine sulfone as an internal standard were added.
The samples were vortex-mixed for 1 minute and filtered with a Millipore filter to remove proteins. Finally, the samples were centrifuged for 70 minutes, at 2000 rpm, at 4 °C. After centrifugation, 90 µL of the supernatant were transferred to a CE-MS vial for analysis.

Analytical settings for the UHPLC-QTOF-MS analysis
The analysis of the samples was accomplished using an UHPLC system (1200 Infinity system, Agilent Technologies, Waldbronn, Germany), coupled to a 6520 QTOF MS (Agilent Technologies) with an ESI ion source. The sample injection volume was set up to 10 μL. The separation was

Analytical settings for the GC-MS analysis
An Agilent GC system (7890A) coupled to a-5975C mass spectrometer (Agilent Technologies) was used to perform metabolite fingerprinting of plasma samples. Briefly, 2 μL of derivatized 4 samples were automatically injected in split mode (ratio 1:10) through a split liner of ultra-inert deactivated glass wool from Agilent. The separation of the compounds was achieved using a pre-column (10 m J&W integrated with Agilent 122-5532G) combined with a GC DB5-MS column (length, 30 m; internal diameter, 0.25 mm; and 0.25 μm film of 95% of dimethyl/5% diphenylpolysiloxane). The flow rate of the carrier gas (helium) was constant at 1 mL/min through the column. The retention time (RT) was locked according to the peak of the internal standard (C18 methyl stearate) at 19.66 minutes. The temperature of the column was initially set at 60 °C for 1 minute, then raised to 10 °C/min to 325 °C, which was maintained for 10 minutes before cooling. The injector and transfer line temperatures were set at 250 °C and 280 °C, respectively. The operating parameters of electronic impact ionization were established as follows: filament source temperature at 230 ° C and electronic ionization energy at 70 eV. Mass spectra were collected in a mass range of 50 to 600 m/z at a scan rate of 2 spectra per second.
Data was acquired using Agilent MSD ChemStation software (Agilent Technologies). To determine the retention rate, a mixture of n-alkanes (C8-C28) dissolved in n-hexane was analyzed before the samples.

Analytical settings for the CE-MS analysis
The analysis was performed using a 7100 capillary electrophoresis (Agilent Technologies)