Distinct metabolomic signature in cerebrospinal fluid in early parkinson's disease: Early Parkinson'S CSF Metabolic Signature

in Movement Disorders (2017)

Objective: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early ... [more ▼]

Objective: The purpose of this study was to profile cerebrospinal fluid (CSF) from early-stage PD patients for disease-related metabolic changes and to determine a robust biomarker signature for early-stage PD diagnosis. Methods: By applying a non-targeted and mass spectrometry-driven approach, we investigated the CSF metabolome of 44 early-stage sporadic PD patients yet without treatment (DeNoPa cohort). We compared all detected metabolite levels with those measured in CSF of 43 age- and gender-matched healthy controls. After this analysis, we validated the results in an independent PD study cohort (T€ubingen cohort). Results: We identified that dehydroascorbic acid levels were significantly lower and fructose, mannose, and threonic acid levels were significantly higher (P <.05) in PD patients when compared with healthy controls. These changes reflect pathological oxidative stress responses, as well as protein glycation/glycosylation reactions in PD. Using a machine learning approach based on logistic regression, we successfully predicted the origin (PD patients vs healthy controls) in a second (n518) as well as in a third and completely independent validation set (n536). The biomarker signature is composed of the three markers—mannose, threonic acid, and fructose—and allows for sample classification with a sensitivity of 0.790 and a specificity of 0.800. Conclusion: We identified PD-specific metabolic changes in CSF that were associated with antioxidative stress response, glycation, and inflammation. Our results disentangle the complexity of the CSF metabolome to unravel metabolome changes related to earlystage PD. The detected biomarkers help understanding PD pathogenesis and can be applied as biomarkers to increase clinical diagnosis accuracy and patient care in early-stage PD. [less ▲]

Method optimization for fecal sample collection and fecal DNA extraction.

in Biopreservation and biobanking (2015), 13(2), 79-93

BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here ... [more ▼]

BACKGROUND: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses. METHODS: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool. Three DNA extraction methods (PerkinElmer MSM I(R), Norgen Biotek All-In-One(R), MoBio PowerMag(R)) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. RESULTS: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 muL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20 degrees C or -80 degrees C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/muL. The protocol was reproducible in terms of DNA yield among different stool aliquots. CONCLUSIONS: We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation. [less ▲]

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

in Biopreservation and biobanking (2014), 12(5), 351-7

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was ... [more ▼]

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. METHODS: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4 degrees C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4 degrees C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. RESULTS: The optimal protocol was a 20-min, 12,000 g centrifugation at 4 degrees C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4 degrees C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. CONCLUSIONS: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4 degrees C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation. [less ▲]