Casey Chamberlain (Presenter)
University of Florida
Bio: Name: Casey A. Chamberlain Organization: University of Florida Occupation: PhD Candidate, Laboratory of Timothy J. Garrett, PhD Education: B.S. Chemistry, University of Florida Current Interests: Metabolomics of the Intestinal Microbiome, Sample Quality
Authorship: Casey A. Chamberlain (1), Christopher Beecher (2), Timothy J. Garrett (1)
(1) University of Florida, Gainesville, FL (2) IROA Technologies, Gainesville, FL
| Short Abstract This study seeks to define global trends in human plasma under various storage conditions to identify biomarkers for sample age and quality. Plasma was obtained through American Red Cross (pooled from approximately 5,000 individuals) as well as from four individual subjects. Analysis by UHPLC-HRMS was conducted using a Thermo Scientific Q Exactive coupled to a Dionex Ultimate 3000 UHPLC System. The effect of butylated hydroxytoluene (BHT) in stabilizing plasma metabolites was also investigated. Many significantly-changing metabolites were identified and evaluated as biomarkers for sample quality. Plasma showed to be largely unstable when left at ambient temperature even for short periods of time. |
Long Abstract
Introduction
The degradation of plasma metabolites is highly-important from both a clinical and research perspective to ensure accurate and thorough analysis of diagnostic and experimental assays; however, little work has been done to fully understand it. Identifying the degradation products of plasma and other biologically relevant samples is critical as these compounds could serve as biomarkers for sample age and quality. This study seeks to identify changes in the metabolic profile of human plasma resulting from exposure to storage at 4°C and ambient temperature (25°C). This work examines these trends in pooled plasma obtained from the American Red Cross as well as in four individual subjects. Also investigated is the usefulness of butylated hydroxytoluene (BHT) for stabilizing plasma under the conditions studied.
Methods
Plasma was divided into identical aliquots and stored at -80°C. At each time point (0, 1, 2, 4, 8 hours), aliquots were pulled from the freezer, allowed to thaw, and conditioned using a thermoblock. Samples were immediately re-frozen after conditioning. At the conclusion of sample collection, all samples within each study were processed simultaneously using our standard 8:1:1 acetonitrile:methanol:acetone metabolite extraction protocol. All analyses were performed using a ThermoScientific Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer coupled to a Dionex Ultimate 3000 UHPLC System. Separation was conducted using reverse phase chromatography following gradient elution (100% water + 0.1% formic acid to 80% acetonitrile). Data were initially processed using Xcalibur Workstation (version 3.0) and RawConverter software. Peak picking was performed using MZmine 2 software, and MetaboAnalyst 3.0 was used for the statistical analysis.
Results
Plasma in the ambient study showed significant global separation after 2-4 hours, providing supporting evidence for the importance of handling samples on ice at all times during typical laboratory procedures as the effects of temperature are likely cumulative. Many compounds, known and unknown, showed distinct patterns over time: increased abundance (likely degradation product), decreased abundance (sensitive to degradation), and relatively constant abundance (resistant to degradation). The changes in abundance experienced by many of these compounds were statistically significant (p<0.05, some as low as 3×10-39), allowing them to potentially serve as sample quality biomarkers. BHT was found to be effective at stabilizing certain metabolites more than others, although a deeper investigation is needed to determine optimal conditions for its use.
Conclusions & Discussion
This study describes that human plasma is a constantly changing chemical matrix and provides evidence for the necessity to characterize its age and related qualities in order to improve global metabolomic studies. Many of the compounds identified in this study may provide a method to ascertain the quality of a specimen and then to remove it from statistical considerations if necessary. This eliminates the potential for inaccurate results due to sample degradation.
References & Acknowledgements:
Acknowledgments
Timothy J. Garrett, PhD
Joy D. Guingab, PhD
Vanessa Y. Rubio
Laurel Meke
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